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Sample records for m1 receptor function

  1. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    Science.gov (United States)

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

  2. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

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    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  3. E1 and M1 strength functions at low energy

    Science.gov (United States)

    Schwengner, Ronald; Massarczyk, Ralph; Bemmerer, Daniel; Beyer, Roland; Junghans, Arnd R.; Kögler, Toni; Rusev, Gencho; Tonchev, Anton P.; Tornow, Werner; Wagner, Andreas

    2017-09-01

    We report photon-scattering experiments using bremsstrahlung at the γELBE facility of Helmholtz-Zentrum Dresden-Rossendorf and using quasi-monoenergetic, polarized γ beams at the HIγS facility of the Triangle Universities Nuclear Laboratory in Durham. To deduce the photoabsorption cross sections at high excitation energy and high level density, unresolved strength in the quasicontinuum of nuclear states has been taken into account. In the analysis of the spectra measured by using bremsstrahlung at γELBE, we perform simulations of statistical γ-ray cascades using the code γDEX to estimate intensities of inelastic transitions to low-lying excited states. Simulated average branching ratios are compared with model-independent branching ratios obtained from spectra measured by using monoenergetic γ beams at HIγS. E1 strength in the energy region of the pygmy dipole resonance is discussed in nuclei around mass 90 and in xenon isotopes. M1 strength in the region of the spin-flip resonance is also considered for xenon isotopes. The dipole strength function of 74Ge deduced from γELBE experiments is compared with the one obtained from experiments at the Oslo Cyclotron Laboratory. The low-energy upbend seen in the Oslo data is interpreted as M1 strength on the basis of shell-model calculations.

  4. E1 and M1 strength functions at low energy

    Directory of Open Access Journals (Sweden)

    Schwengner Ronald

    2017-01-01

    Full Text Available We report photon-scattering experiments using bremsstrahlung at the γELBE facility of Helmholtz-Zentrum Dresden-Rossendorf and using quasi-monoenergetic, polarized γ beams at the HIγS facility of the Triangle Universities Nuclear Laboratory in Durham. To deduce the photoabsorption cross sections at high excitation energy and high level density, unresolved strength in the quasicontinuum of nuclear states has been taken into account. In the analysis of the spectra measured by using bremsstrahlung at γELBE, we perform simulations of statistical γ-ray cascades using the code γDEX to estimate intensities of inelastic transitions to low-lying excited states. Simulated average branching ratios are compared with model-independent branching ratios obtained from spectra measured by using monoenergetic γ beams at HIγS. E1 strength in the energy region of the pygmy dipole resonance is discussed in nuclei around mass 90 and in xenon isotopes. M1 strength in the region of the spin-flip resonance is also considered for xenon isotopes. The dipole strength function of 74Ge deduced from γELBE experiments is compared with the one obtained from experiments at the Oslo Cyclotron Laboratory. The low-energy upbend seen in the Oslo data is interpreted as M1 strength on the basis of shell-model calculations.

  5. Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

    DEFF Research Database (Denmark)

    Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig

    2010-01-01

    substituted for cocaine and enhanced its discriminative stimulus. Conversely, muscarinic agonists blunted cocaine discrimination and abolished cocaine self-administration with varying effects on food-maintained behavior. Specifically, increasing selectivity for the M(1) subtype (oxotremorine ...'s abuse-related effects, whereas non-M(1)/M(4) receptors probably contribute to undesirable effects of muscarinic stimulation. These data provide the first demonstration of anticocaine effects of systemically applied, M(1) receptor agonists and suggest the possibility of a new approach to pharmacotherapy...

  6. Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

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    Mathew Jobin

    2009-04-01

    Full Text Available Abstract Acetylcholine (ACh, the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS. Previous reports from our laboratory on streptozotocin (STZ induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax and affinity (Kd of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

  7. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys.

    Science.gov (United States)

    Wu, Wei; Saunders, Richard C; Mishkin, Mortimer; Turchi, Janita

    2012-07-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. Published by Elsevier Inc.

  8. Alterations of M1 and M4 acetylcholine receptors in the genetically dystonic (dtsz) hamster and moderate antidystonic efficacy of M1 and M4 anticholinergics.

    Science.gov (United States)

    Hamann, Melanie; Plank, Jagoda; Richter, Franziska; Bode, Christoph; Smiljanic, Sinisa; Creed, Meaghan; Nobrega, José N; Richter, Angelika

    2017-08-15

    Striatal cholinergic dysfunction has been suggested to play a critical role in the pathophysiology of dystonia. In the dt sz hamster, a phenotypic model of paroxysmal dystonia, M1 antagonists exerted moderate antidystonic efficacy after acute systemic administration. In the present study, we examined the effects of the M4 preferring antagonist tropicamid and whether long-term systemic or acute intrastriatal injections of the M1 preferring antagonist trihexyphenidyl are more effective in mutant hamsters. Furthermore, M1 and M4 receptors were analyzed by autoradiography and immunohistochemistry. Tropicamide retarded the onset of dystonic attacks, as previously observed after acute systemic administration of trihexyphenidyl. Combined systemic administration of trihexyphenidyl (30mg/kg) and tropicamide (15mg/kg) reduced the severity in acute trials and delayed the onset of dystonia during long-term treatment. In contrast, acute striatal microinjections of trihexyphenidyl, tropicamid or the positive allosteric M4 receptor modulator VU0152100 did not exert significant effects. Receptor analyses revealed changes of M1 receptors in the dorsomedial striatum, suggesting that the cholinergic system is involved in abnormal striatal plasticity in dt sz hamsters, but the pharmacological data argue against a crucial role on the phenotype in this animal model. However, antidystonic effects of tropicamide after systemic administration point to a novel therapeutic potential of M4 preferring anticholinergics for the treatment of dystonia. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. The muscarinic M1/M4 receptor agonist xanomeline exhibits antipsychotic-like activity in Cebus apella monkeys

    DEFF Research Database (Denmark)

    Andersen, Maibritt B; Fink-Jensen, Anders; Peacock, Linda

    2003-01-01

    (EPS) at therapeutically relevant doses. In the present study, we examined whether the xanomeline-induced functional dopamine antagonism found in rodent studies could also be observed in nonhuman primates. In addition, we studied whether the lack of EPS observed in rodents also applies to primates......Xanomeline is a muscarinic M(1)/M(4) preferring receptor agonist with little or no affinity for dopamine receptors. The compound reduces psychotic-like symptoms in patients with Alzheimer's disease and exhibits an antipsychotic-like profile in rodents without inducing extrapyramidal side effects...

  10. Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

    DEFF Research Database (Denmark)

    Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig

    2010-01-01

    Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus...... for cocaine addiction....

  11. Current status of muscarinic M1 and M4 receptors as drug targets for neurodegenerative diseases.

    Science.gov (United States)

    Felder, Christian C; Goldsmith, Paul J; Jackson, Kimberley; Sanger, Helen E; Evans, David A; Mogg, Adrian J; Broad, Lisa M

    2018-01-25

    The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders. Copyright © 2018. Published by Elsevier Ltd.

  12. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

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    Joseph, Lauren; Thomsen, Morgane

    2017-06-30

    Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Co-expression of two subtypes of melatonin receptor on rat M1-type intrinsically photosensitive retinal ganglion cells.

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    Wen-Long Sheng

    Full Text Available Intrinsically photosensitive retinal ganglion cells (ipRGCs are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

  14. Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

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    Eva Šantrůčková

    Full Text Available Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

  15. Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

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    Galloway, Claire R; Lebois, Evan P; Shagarabi, Shezza L; Hernandez, Norma A; Manns, Joseph R

    2014-01-01

    Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

  16. The muscarinic M1/M4 receptor agonist xanomeline exhibits antipsychotic-like activity in Cebus apella monkeys

    DEFF Research Database (Denmark)

    Andersen, Maibritt B; Fink-Jensen, Anders; Peacock, Linda

    2003-01-01

    Xanomeline is a muscarinic M(1)/M(4) preferring receptor agonist with little or no affinity for dopamine receptors. The compound reduces psychotic-like symptoms in patients with Alzheimer's disease and exhibits an antipsychotic-like profile in rodents without inducing extrapyramidal side effects ...

  17. Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

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    Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

    2017-01-01

    Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

  18. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Scherer, N.M.; Nathanson, N.M.

    1990-01-01

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

  19. Immunocytochemical demonstration of M1 muscarinic acetylcholine receptors at the presynaptic and postsynaptic membranes of rat diaphragm endplates

    Czech Academy of Sciences Publication Activity Database

    Malomouzh, A. I.; Arkhipova, S. S.; Nikolsky, E. E.; Vyskočil, František

    2011-01-01

    Roč. 60, č. 1 (2011), s. 185-188 ISSN 0862-8408 R&D Projects: GA AV ČR(CZ) IAA500110905; GA ČR GA202/09/0806 Institutional research plan: CEZ:AV0Z50110509 Keywords : skeletal muscle * M1 muscarinic receptor Subject RIV: ED - Physiology Impact factor: 1.555, year: 2011

  20. Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Tuček, Stanislav; El-Fakahany, E. E.

    2002-01-01

    Roč. 301, č. 3 (2002), s. 1033-1041 ISSN 0022-3565 R&D Projects: GA ČR GP305/01/D119 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * M(1) muscarinic acetylcholine receptor Subject RIV: FH - Neurology Impact factor: 3.991, year: 2002

  1. The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    International Nuclear Information System (INIS)

    Mei, Lin.

    1989-01-01

    The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

  2. Cyclic GMP-dependent protein kinase II is necessary for macrophage M1 polarization and phagocytosis via toll-like receptor 2.

    Science.gov (United States)

    Liao, Wei-Ting; You, Huey-Ling; Li, Changgui; Chang, Jan-Gowth; Chang, Shun-Jen; Chen, Chung-Jen

    2015-05-01

    Cyclic GMP-dependent protein kinase II (cGKII; PRKG2) phosphorylates a variety of biological targets and has been identified as a gout-susceptible gene. However, the regulatory role of cGKII in triggering gout disease has yet to be clarified. Thus, we plan to explore the specific function of cGKII in macrophages related to gout disease. By using cGKII gene knockdown method, we detected macrophage M1/M2 polarization, phagocytosis, and their responses to stimulation by monosodium urate (MSU). cGKII was highly expressed in M1 phenotype, but not in M2, and cGKII knockdown significantly inhibited macrophage M1 polarization by decreasing M1 chemokine markers (CXCL10 and CCL2) and downregulating phagocytosis function. We further identified that cGKII-associated phagocytosis was mediated by upregulating toll-like receptor 2 (TLR2) expression, but not by TLR4. Mimicking gout condition by MSU treatments, we found that MSU alone induced cGKII and TLR2 expression with increased M1 polarization markers and phagocytosis activity. It means that cGKII knockdown significantly inhibited this MSU-induced cGKII-TLR2-phagocytosis axis. Our study showed that cGKII plays a key role in M1 polarization, especially in TLR2-mediated phagocytosis under MSU exposure. The findings provide evidence for the possible role of cGKII as an inflammation exciter in gout disease. Gout-susceptible gene cGKII is necessary for macrophage M1 polarization. cGKII regulates M1 phagocytosis function via TLR2. Monosodium urate treatments increase cGKII expression and related function. This study reveals the role of cGKII in enhancing gouty inflammatory responses.

  3. Presynaptic M1 muscarinic receptor modulates spontaneous release of acetylcholine from rat basal forearm slices

    International Nuclear Information System (INIS)

    Suzuki, T.; Fujimoto, LK.; Oohata, H.; Kawashima, K.

    1988-01-01

    Spontaneous release of (ACh) from rat basal forebrain slices in the presence of cholinesterase inhibitor was directly determined using a specific radioimmunoassay for ACh. The release was calcium dependent. A consistent amount of ACh release was observed throughout the experiment. Atropine (10- 8 to 10- 5 M) and pirenzepine (10- 7 to 10- 5 M) enhanced spontaneous ACh release. These findings indicate the presence of an M 1 muscarenic autoreceptor that modulates spontaneous release of ACh in the rat forebrain

  4. Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid beta(1-42)

    Czech Academy of Sciences Publication Activity Database

    Janíčková, Helena; Rudajev, Vladimír; Zimčík, Pavel; Jakubík, Jan; Tanila, H.; El-Fakahany, E. E.; Doležal, Vladimír

    2013-01-01

    Roč. 67, April (2013), s. 272-283 ISSN 0028-3908 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) 7E10060 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * muscarinic receptors * G-proteins Subject RIV: ED - Physiology Impact factor: 4.819, year: 2013

  5. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

    Czech Academy of Sciences Publication Activity Database

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, E. E.; Jakubík, Jan

    2015-01-01

    Roč. 97, Jul 2015 (2015), s. 27-39 ISSN 1043-6618 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * atypical agonists * xanomeline * activation mechanism Subject RIV: ED - Physiology Impact factor: 4.816, year: 2015

  6. Activation of multiple G-proteins by muscarinic M1 and M2 receptors

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

    2006-01-01

    Roč. 27, č. S1 (2006), s. 404-404 ISSN 1671-4083. [World Congress of Pharmacology /15./. 02.07.2006-07.07.2006, Beijing] R&D Projects: GA ČR(CZ) GP305/05/P209; GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * multiple G-protein coupling Subject RIV: ED - Physiology

  7. Involvement of Striatal Cholinergic Interneurons and M1 and M4 Muscarinic Receptors in Motor Symptoms of Parkinson's Disease.

    Science.gov (United States)

    Ztaou, Samira; Maurice, Nicolas; Camon, Jeremy; Guiraudie-Capraz, Gaëlle; Kerkerian-Le Goff, Lydia; Beurrier, Corinne; Liberge, Martine; Amalric, Marianne

    2016-08-31

    Over the last decade, striatal cholinergic interneurons (ChIs) have reemerged as key actors in the pathophysiology of basal-ganglia-related movement disorders. However, the mechanisms involved are still unclear. In this study, we address the role of ChI activity in the expression of parkinsonian-like motor deficits in a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion model using optogenetic and pharmacological approaches. Dorsal striatal photoinhibition of ChIs in lesioned ChAT(cre/cre) mice expressing halorhodopsin in ChIs reduces akinesia, bradykinesia, and sensorimotor neglect. Muscarinic acetylcholine receptor (mAChR) blockade by scopolamine produces similar anti-parkinsonian effects. To decipher which of the mAChR subtypes provides these beneficial effects, systemic and intrastriatal administration of the selective M1 and M4 mAChR antagonists telenzepine and tropicamide, respectively, were tested in the same model of Parkinson's disease. The two compounds alleviate 6-OHDA lesion-induced motor deficits. Telenzepine produces its beneficial effects by blocking postsynaptic M1 mAChRs expressed on medium spiny neurons (MSNs) at the origin of the indirect striatopallidal and direct striatonigral pathways. The anti-parkinsonian effects of tropicamide were almost completely abolished in mutant lesioned mice that lack M4 mAChRs specifically in dopamine D1-receptor-expressing neurons, suggesting that postsynaptic M4 mAChRs expressed on direct MSNs mediate the antiakinetic action of tropicamide. The present results show that altered cholinergic transmission via M1 and M4 mAChRs of the dorsal striatum plays a pivotal role in the occurrence of motor symptoms in Parkinson's disease. The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinson's disease. Here, using optogenetic and pharmacological approaches, we investigated the involvement of striatal

  8. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1

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    Federica Raggi

    2017-09-01

    Full Text Available Macrophages (Mf are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1 and alternatively activated (anti-inflammatory M2 subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.

  9. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1

    Science.gov (United States)

    Raggi, Federica; Pelassa, Simone; Pierobon, Daniele; Penco, Federica; Gattorno, Marco; Novelli, Francesco; Eva, Alessandra; Varesio, Luigi; Giovarelli, Mirella; Bosco, Maria Carla

    2017-01-01

    Macrophages (Mf) are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1) and alternatively activated (anti-inflammatory M2) subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM)-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment. PMID:28936211

  10. The selective positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning and memory deficits in the Tg2576 Alzheimer's disease mouse model.

    Science.gov (United States)

    Puri, Vanita; Wang, Xiaohai; Vardigan, Joshua D; Kuduk, Scott D; Uslaner, Jason M

    2015-01-01

    We have recently shown that the M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance in rodents and non-human primates administered the muscarinic receptor antagonist scopolamine. The purpose of the present experiments was to characterize the effects of PQCA in a model more relevant to the disease pathology of Alzheimer's disease. Tg2576 transgenic mice that have elevated Aβ were tested in the novel object recognition task to characterize recognition memory as a function of age and treatment with the PQCA. The effects of PQCA were compared to the acetylcholinesterase inhibitor donepezil, the standard of care for Alzheimer's disease. In addition, the effect of co-administering PQCA and donepezil was evaluated. Aged Tg2576 mice demonstrated a deficit in recognition memory that was significantly attenuated by PQCA. The positive control donepezil also reversed the deficit. Furthermore, doses of PQCA and donepezil that were inactive on their own were found to improve recognition memory when given together. These studies suggest that M1 muscarinic receptor positive allosteric modulation can ameliorate memory deficits in disease relevant models of Alzheimer's disease. These data, combined with our previous findings demonstrating PQCA improves scopolamine-induced cognitive deficits in both rodents and non-human primates, suggest that M1 positive allosteric modulators have therapeutic potential for the treatment of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Muscarinic M1 receptor inhibition reduces gastroduodenal bicarbonate secretion and promotes gastric prostaglandin E2 synthesis in healthy volunteers

    DEFF Research Database (Denmark)

    Mertz-Nielsen, A; Hillingsø, Jens; Eskerod, O

    1995-01-01

    stimulated gastric and basal duodenal bicarbonate secretion by about 50% (p basal and vagally stimulated PGE2 output increased significantly (p ...The selective muscarinic M1 receptor antagonist, pirenzepine, considerably stimulates duodenal mucosal bicarbonate secretion in the rat and increases gastric luminal release of prostaglandin E2 (PGE2) in humans. This study, therefore, looked at the effect of pirenzepine on bicarbonate secretion...... sham feeding and acid exposure (HCl 0.1 M; 20 ml; 5 min) of the duodenal bulb increased mucosal bicarbonate secretion from 191 (14) mumol/cm x h to 266 (27) mumol/cm x h (p basal and vagally...

  12. Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

    Science.gov (United States)

    Mannan Baig, Abdul; Khan, Naveed A; Effendi, Vardah; Rana, Zohaib; Ahmad, H R; Abbas, Farhat

    2017-01-01

    Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

  13. Effects of muscarinic M1 and M4 acetylcholine receptor stimulation on extinction and reinstatement of cocaine seeking in male mice, independent of extinction learning.

    Science.gov (United States)

    Stoll, Kevin; Hart, Rachel; Lindsley, Craig W; Thomsen, Morgane

    2018-03-01

    Stimulating muscarinic M 1 /M 4 receptors can blunt reinforcing and other effects of cocaine. A hallmark of addiction is continued drug seeking/craving after abstinence and relapse. We tested whether stimulating M 1 and/or M 4 receptors could facilitate extinction of cocaine seeking, and whether this was mediated via memory consolidation. Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1 mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding extinguished to ≤30% of cocaine-reinforced responding. Immediately after each extinction session, mice received saline, the M 1 /M 4 receptor-preferring agonist xanomeline, the M 1 receptor-selective allosteric agonist VU0357017, the M 4 receptor-selective positive allosteric modulator VU0152100, or VU0357017 + VU0152100. In additional experiments, xanomeline was administered delayed after the session or in the home cage before extinction training began. In the latter group, reinstatement of responding by a 10-mg/kg cocaine injection was also tested. Stimulating M 1  + M 4 receptors significantly expedited extinction from 17.2 sessions to 8.3 using xanomeline or 7.8 using VU0357017 + VU0152100. VU0357017 alone and VU0152100 alone did not significantly modify rates of extinction (12.6 and 14.6 sessions). The effect of xanomeline was fully preserved when administered delayed after or unpaired from extinction sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. These findings show that M 1 /M 4 receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests that M 1 /M 4 receptor stimulation modulated or reversed some neurochemical effects of cocaine exposure.

  14. Effects of agonist efficacy on desensitization of phosphoinositide hydrolysis mediated by m1 and m3 muscarinic receptors expressed in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Hu, J.; Wang, S.Z.; el-Fakahany, E.E.

    1991-01-01

    Muscarinic receptor agonist-induced desensitization of phosphoinositide (PI) hydrolysis and loss of receptors were studied in Chinese hamster ovary (CHO) cells transfected with the m1 and m3 muscarinic receptor genes. Long-term exposure to the full agonist carbamylcholine (CBC) resulted in a time-dependent attenuation of the maximal PI response and a decrease in agonist potency. This desensitization was accompanied by a parallel loss of maximal ligand binding without an alteration of the binding affinity. The time course of both receptor desensitization and down-regulation was similar in m1 and m3 CHO cells. The PI response to the partial agonist McN-A-343 (McN) in m1 cells was more sensitive to desensitization by CBC than the response to the latter agonist, and this desensitization was faster than receptor down-regulation. Desensitization of the PI response to McN was reflected as a decrease in the maximal response without a marked change in potency. McN induced slow desensitization of the PI response to CBC but a much faster desensitization of its own response. Our data provide evidence that although muscarinic agonist-induced desensitization of PI hydrolysis in CHO cells is due mainly to loss of receptors, there are other important factors which play a role in this process, e.g., receptor-effector uncoupling. The relative contribution of these different mechanisms depends on the efficacy of the agonists used for the receptor desensitization and activation steps

  15. Disturbed mitochondrial function restricts glutamate uptake in the human Müller glia cell line, MIO-M1

    DEFF Research Database (Denmark)

    Vohra, Rupali; Gurubaran, Iswariyaraja Sridevi; Henriksen, Ulrik

    2017-01-01

    Using the human Müller cell line, MIO-M1, the aim was to study the impact of mitochondrial inhibition in Müller glia through antimycin A treatment. MIO-M1 cell survival, levels of released lactate, mitochondrial function, and glutamate uptake were studied in response to mitochondrial inhibition...... and glucose restriction. Lactate release decreased in response to glucose restriction. Combined glucose restriction and blocked mitochondrial activity decreased survival and caused collapse of the respiratory chain measured by oxygen consumption rate and extracellular acidification rate. Mitochondrial...... inhibition caused impaired glutamate uptake and decreased mRNA expression of the glutamate transporter, EAAT1. Over all, we show important roles of mitochondrial activity in MIO-M1 cell function and survival....

  16. Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors.

    Directory of Open Access Journals (Sweden)

    Mohammad Harun-Or-Rashid

    Full Text Available Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR system and extracellular signal-regulated kinase (ERK signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173 EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2, EGFR-inhibitor (AG1478, EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001, consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in

  17. Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

    Science.gov (United States)

    Lee, Ok Ran; Cho, Hyung-Taeg

    2012-12-01

    Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

  18. Trichoderma asperelloides Spores Downregulate dectin1/2 and TLR2 Receptors of Mice Macrophages and Decrease Candida parapsilosis Phagocytosis Independent of the M1/M2 Polarization

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    Andréa G. dos Santos

    2017-09-01

    Full Text Available The intensive use of pesticides to control pests in agriculture has promoted several issues relating to environment. As chemical pesticides remain controversial, biocontrol agents originating from fungi could be an alternative. Among them, we highlight biocontrol agents derived from the fungi genus Trichoderma, which have been documented in limiting the growth of other phytopathogenic fungus in the roots and leaves of several plant species. An important member of this genus is Trichoderma asperelloides, whose biocontrol agents have been used to promote plant growth while also treating soil diseases caused by microorganisms in both greenhouses and outdoor crops. To evaluate the safety of fungal biological agents for human health, tests to detect potentially adverse effects, such as allergenicity, toxicity, infectivity and pathogenicity, are crucial. In addition, identifying possible immunomodulating properties of fungal biocontrol agents merits further investigation. Thus, the aim of this study was to evaluate the effects of T. asperelloides spores in the internalization of Candida parapsilosis yeast by mice phagocytes, in order to elucidate the cellular and molecular mechanism of this interaction, as a model to understand possible in vivo effects of this fungus. For this, mice were exposed to a fungal spore suspension through-intraperitoneal injection, euthanized and cells from the peripheral blood and peritoneal cavity were collected for functional, quantitative and phenotypic analysis, throughout analysis of membrane receptors gene expression, phagocytosis ability and cells immunophenotyping M1 (CCR7 and CD86 and M2 (CCR2 and CD206. Our analyses showed that phagocytes exposed to fungal spores had reduced phagocytic capacity, as well as a decrease in the quantity of neutrophils and monocytes in the peripheral blood and peritoneal cavity. Moreover, macrophages exposed to T. asperelloides spores did not display the phenotypic profile M1/M2, and

  19. In situ hybridization on the change of m1 receptor mRNA in different brain areas of aged rats and the effect of Yin tonic Zhimu studied

    International Nuclear Information System (INIS)

    Hu Yaer; Xia Zongqin; Yi Ningyu

    1996-01-01

    The change of gene expression of m1 receptors in different brain areas of aged rats and the effects of water extract of the Yin tonic Zhimu and its active principle ZMS was studied. In situ hybridization using 35 S-labelled m1 and m2 probes and analysis of the autoradiographs using a computerized image-analyzer was selected. The grain density of m1 mRNA in striatum was significantly lowered in old rats as compared with young rats (decreased by 12.26 +- 3.60, P<0.01). Long-term oral administration of ZMS, the active principle of Yin tonic Zhimu but not the water extraction of Zhimu, elevated the m1 mRNA in striatum of aged rats (increased by 15.71 +- 3.27, P<0.01). Neither significant change of the grain density of m1 mRNA in old rats nor significant effect of Zhimu or ZMS was observed in cerebral cortex and hippocampus. The m1 mRNA level in striatum is decreased in aged rats and ZMS is able to elevate it

  20. Influence of the projection of BCS functions on the M1 and E2 transitions in rare earths

    International Nuclear Information System (INIS)

    Fellah, M.; Hammann, T.F.

    1975-01-01

    E2 and M1 transition probabilities for odd-mass rare earth nuclei, have been calculated using both the usual BCS wave functions and the strict particle conserving, projected BCS functions. The blocking effect has been exactly and systematically taken into account. The influence of the Coriolis interaction has been studied using the first order perturbation theory. Allowance has been made for the β and γ vibrations. The unphysical effects, due to particle fluctuation in the BCS theory, are not always negligible, but are in most cases, less important than the Coriolis effect [fr

  1. Effects of muscarinic M1 and M4 acetylcholine receptor stimulation on extinction and reinstatement of cocaine seeking in male mice, independent of extinction learning

    DEFF Research Database (Denmark)

    Stoll, Kevin; Hart, Rachel; Lindsley, Craig W

    2017-01-01

    seeking, and whether this was mediated via memory consolidation. METHODS: Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1 mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding...... sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. CONCLUSIONS: These findings show that M1/M4 receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests...

  2. Muscarinic acetylcholine receptor subtypes: localization and structure/function

    DEFF Research Database (Denmark)

    Brann, M R; Ellis, J; Jørgensen, H

    1993-01-01

    Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues...... are described, as well as the implied structures of these functional domains....

  3. Effect of insulin analogues on insulin/IGF1 hybrid receptors: increased activation by glargine but not by its metabolites M1 and M2.

    Directory of Open Access Journals (Sweden)

    Cécile Pierre-Eugene

    Full Text Available BACKGROUND: In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R, present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. METHODOLOGY: To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP(3 production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP(3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. RESULTS: Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP(3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP(3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. CONCLUSION: Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in

  4. Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

    Science.gov (United States)

    Hasebe, Masaharu

    2016-01-01

    The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

  5. Autoradiography of H-3-pirenzepine and H-3-AFDX-384 in Mouse Brain Regions: Possible Insights into M-1, M-2, and M-4 Muscarinic Receptors Distribution

    Czech Academy of Sciences Publication Activity Database

    Valuskova, P.; Farar, V.; Forczek, Sándor; Křížová, I.; Mysliveček, J.

    2018-01-01

    Roč. 9, FEB 20 (2018), č. článku 124. ISSN 1663-9812 Institutional support: RVO:61389030 Keywords : 3 h-afdx-384 * 3 H-pirenzepine * 3 h-qnb * Autoradiography * M muscarinic receptor 1 * M muscarinic receptor 2 * M muscarinic receptor 4 Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.400, year: 2016

  6. Mediator-dependent Nuclear Receptor Functions

    Science.gov (United States)

    Chen, Wei; Roeder, Robert

    2011-01-01

    As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

  7. Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

    Directory of Open Access Journals (Sweden)

    Karina Sánchez-Reyes

    2014-01-01

    Full Text Available Cervical cancer (CC is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated and M2 (alternatively activated. Macrophage polarization exerts profound effects on the Toll-like receptor (TLR profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

  8. Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

    Science.gov (United States)

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

    2016-06-23

    The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors

  9. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber)

    DEFF Research Database (Denmark)

    Jørgensen, Kristine B.; Krogh-Jensen, Karen; Pickering, Darryl S

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1–5 of the naked mole-rat were compared to that of the house mouse (Mus...... musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies...

  10. Conformational regulation of urokinase receptor function

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Jacobsen, Benedikte; Kriegbaum, Mette C

    2011-01-01

    PA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized...

  11. Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

    2015-01-01

    Roč. 40, č. 10 (2015), s. 2068-2070 ISSN 0364-3190 R&D Projects: GA ČR(CZ) GA14-05696S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) 7E10060 Institutional support: RVO:67985823 Keywords : muscarinic receptors * agonist binding * cholesterol * G-proteins * signal transduction * cAMP synthesis Subject RIV: FH - Neurology Impact factor: 2.472, year: 2015

  12. Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

    2006-01-01

    Roč. 70, č. 2 (2006), s. 656-666 ISSN 0026-895X R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * muscarinic receptors * G-protein activation Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.469, year: 2006

  13. Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

    Czech Academy of Sciences Publication Activity Database

    Malomouzh, A. I.; Mukhtarov, M. R.; Nikolsky, E. E.; Vyskočil, František

    2007-01-01

    Roč. 102, č. 6 (2007), s. 2110-2117 ISSN 0022-3042 R&D Projects: GA AV ČR(CZ) IAA5011411; GA MŠk(CZ) LC554 Grant - others:-(RU) 112.0/001/481 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * NO synthese Subject RIV: ED - Physiology Impact factor: 4.451, year: 2007

  14. Functional reconstitution of the glycine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Calvo, M.; Ruiz-Gomez, A.; Vazquez, J.; Morato, E.; Valdivieso, F.; Mayor, F. Jr. (Universidad Autonoma de Madrid (Spain))

    1989-07-25

    The functional reconstitution of the chloride channel coupled glycine receptor is described. Glycine receptors were purified from the cholate extract of rat spinal cord membranes by affinity chromatography and incorporated into phospholipid vesicles by the addition of phosphatidylcholine and removal of detergent by gel filtration. The reconstituted vesicles showed the same polypeptide composition as the purified receptor. The pharmacological characteristics of the glycine receptor were also preserved in the proteoliposomes, as demonstrated by the displacement of ({sup 3}H)strychnine binding by several glycinergic ligands and by photoaffinity labeling experiments. In order to observe functional responses (i.e., specific agonist-induced anion translocation), the authors have developed an assay based on the fluorescence quenching of an anion-sensitive entrapped probe, SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). Reconstituted vesicles were loaded with the fluorescent probe during a freeze-thaw-sonication cycle in the presence of added liposomes containing cholesterol. In such a reconstituted system, glycine receptor agonists are able to increase the rate of anion influx into the vesicles. The action of agonists is blocked by the simultaneous presence of strychnine or other glycine antagonists. The results show that the purified 48,000- and 58,000-dalton polypeptides reconstituted into phospholipid vesicles can bind ligands and promote specific ion translocation in a way similar to the glycine receptor in its native environment.

  15. Pharmacology and function of melatonin receptors

    International Nuclear Information System (INIS)

    Dubocovich, M.L.

    1988-01-01

    The hormone melatonin is secreted primarily from the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone, through an action in the brain, appears to be involved in the regulation of various neural and endocrine processes that are cued by the daily change in photoperiod. This article reviews the pharmacological characteristics and function of melatonin receptors in the central nervous system, and the role of melatonin in mediating physiological functions in mammals. Melatonin and melatonin agonists, at picomolar concentrations, inhibit the release of dopamine from retina through activation of a site that is pharmacologically different from a serotonin receptor. These inhibitory effects are antagonized by the novel melatonin receptor antagonist luzindole (N-0774), which suggests that melatonin activates a presynaptic melatonin receptor. In chicken and rabbit retina, the pharmacological characteristics of the presynaptic melatonin receptor and the site labeled by 2-[125I]iodomelatonin are identical. It is proposed that 2-[125I]iodomelatonin binding sites (e.g., chicken brain) that possess the pharmacological characteristics of the retinal melatonin receptor site (order of affinities: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-di-chloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin greater than N-acetyltryptamine greater than or equal to luzindole greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine much greater than 5-hydroxytryptamine) be classified as ML-1 (melatonin 1). The 2-[125I]iodomelatonin binding site of hamster brain membranes possesses different binding and pharmacological characteristics from the retinal melatonin receptor site and should be classified as ML-2. 64 references

  16. Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type, which mediates inhibition of tumour immune-evasion via the Toll-like receptor 2 pathway.

    Science.gov (United States)

    Liu, Yi; Zhang, Lingyun; Zhu, Xiangxiang; Wang, Yuehua; Liu, WenWei; Gong, Wei

    2015-11-01

    Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti-cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin-6 (IL-6), IL-12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr-1(+) CD11b(+) monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll-like receptor 2 (TLR2) signal of Gr-1(+) CD11b(+) MDSCs and increased their M1-type macrophage characteristics, such as producing IL-12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr-1(+) CD11b(+) MDSCs by pAbM treatment had less ability to convert the CD4(+) CD25(-) cells into CD4(+) CD25(+) phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti-tumour effect of pAbM was dependent on Gr-1(+ ) CD11b(+) monocytes, nether CD8(+) T cells nor CD4(+) T cells. In addition to, pAbM did not inhibit tumour growth in TLR2(-/-) mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti-tumour effects through the opposite of the suppressive function of Gr-1(+) CD11b(+) MDSCs. © 2015 John Wiley & Sons Ltd.

  17. Functional Validation of Heteromeric Kainate Receptor Models.

    Science.gov (United States)

    Paramo, Teresa; Brown, Patricia M G E; Musgaard, Maria; Bowie, Derek; Biggin, Philip C

    2017-11-21

    Kainate receptors require the presence of external ions for gating. Most work thus far has been performed on homomeric GluK2 but, in vivo, kainate receptors are likely heterotetramers. Agonists bind to the ligand-binding domain (LBD) which is arranged as a dimer of dimers as exemplified in homomeric structures, but no high-resolution structure currently exists of heteromeric kainate receptors. In a full-length heterotetramer, the LBDs could potentially be arranged either as a GluK2 homomer alongside a GluK5 homomer or as two GluK2/K5 heterodimers. We have constructed models of the LBD dimers based on the GluK2 LBD crystal structures and investigated their stability with molecular dynamics simulations. We have then used the models to make predictions about the functional behavior of the full-length GluK2/K5 receptor, which we confirmed via electrophysiological recordings. A key prediction and observation is that lithium ions bind to the dimer interface of GluK2/K5 heteromers and slow their desensitization. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Kupffer cell complement receptor clearance function and host defense.

    Science.gov (United States)

    Loegering, D J

    1986-01-01

    Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

  19. Evaluation of stereoisomers of 4-fluoroalkyl analogues of 3-quinuclidinyl benzilate in in vivo competition studies for the M1, M2, and M3 muscarinic receptor subtypes in brain

    International Nuclear Information System (INIS)

    Kiesewetter, Dale O.; Eckelman, William C.; Jaetae, Lee; Paik, Chang H.; Park, Seok G.

    1995-01-01

    To develop a subtype selective muscarinic acetylcholine receptor (mAChR) antagonist for PET, fluorine-19 labeled alkyl analogues of quinuclidinyl benzilate (QNB) were synthesized by stereoselective reactions. To investigate these analogues for tissue subtype specificity, in vivo competitive binding studies were performed in rat brain using (R)-3-quinuclidinyl (R)-4-[ 125 I]Iodobenzilate (IQNB). Five, fifty, or five-hundred nmol of the non-radioactive ligands were coinjected intravenously with 8 pmol of the radioligand. Cold (R,R)-IQNB blocked (R,R)-[ 125 I]IQNB in a dose-dependent manner, without showing regional specificity. For the (R,S)-fluoromethyl, -fluoroethyl, and -fluoropropyl derivatives, a higher percent blockade was seen at 5 and 50 nmol levels in M2 predominant tissues (medulla, pons, and cerebellum) than in M1 predominant tissues (cortex, striatum and hippocampus). The blockade pattern of the radioligand also correlated qualitatively with the percentage of M2 receptors in the region. The S-quinuclidinyl analogues showed M2 selectivity but less efficient blockade of the radioligand, indicating lower affinities. Radioligand bound to the medulla was inversely correlated to the M2 relative binding affinity of the fluoroalkyl analogues. These results indicate that the nonradioactive ligand blocks the radioligand based on the affinity of the nonradioactive ligand for a particular receptor subtype compared to the affinity of the radioligand for the same receptor subtype. Of the seven compounds evaluated, (R,S)-fluoromethyl-QNB appears to show the most selectivity for the M2 subtypes in competition studies in vivo

  20. Dynamic mobility of functional GABAA receptors at inhibitory synapses.

    Science.gov (United States)

    Thomas, Philip; Mortensen, Martin; Hosie, Alastair M; Smart, Trevor G

    2005-07-01

    Importing functional GABAA receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABAA receptor alpha1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABAA receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABAA receptors at inhibitory synapses and a means to control synaptic efficacy.

  1. Compartmentalization of B-cell antigen receptor functions

    NARCIS (Netherlands)

    Lankester, A. C.; van Lier, R. A.

    1996-01-01

    Receptor tyrosine kinases (RTK), like the PDGF-receptor, translate information from the extracellular environment into cytoplasmic signals that regulate a spectrum of cellular functions. RTK molecules consist of ligand binding extracellular domains, cytoplasmic kinase domains and tyrosine

  2. Functionally heterogenous ryanodine receptors in avian cerebellum.

    Science.gov (United States)

    Sierralta, J; Fill, M; Suárez-Isla, B A

    1996-07-19

    The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

  3. Antioxidant Functions of the Aryl Hydrocarbon Receptor

    Directory of Open Access Journals (Sweden)

    Cornelia Dietrich

    2016-01-01

    Full Text Available The aryl hydrocarbon receptor (AhR is a transcription factor belonging to the basic helix-loop-helix/PER-ARNT-SIM family. It is activated by a variety of ligands, such as environmental contaminants like polycyclic aromatic hydrocarbons or dioxins, but also by naturally occurring compounds and endogenous ligands. Binding of the ligand leads to dimerization of the AhR with aryl hydrocarbon receptor nuclear translocator (ARNT and transcriptional activation of several xenobiotic phase I and phase II metabolizing enzymes. It is generally accepted that the toxic responses of polycyclic aromatic hydrocarbons, dioxins, and structurally related compounds are mediated by activation of the AhR. A multitude of studies indicate that the AhR operates beyond xenobiotic metabolism and exerts pleiotropic functions. Increasing evidence points to a protective role of the AhR against carcinogenesis and oxidative stress. Herein, I will highlight data demonstrating a causal role of the AhR in the antioxidant response and present novel findings on potential AhR-mediated antioxidative mechanisms.

  4. Sweet Taste Receptor Signaling Network: Possible Implication for Cognitive Functioning

    Directory of Open Access Journals (Sweden)

    Menizibeya O. Welcome

    2015-01-01

    Full Text Available Sweet taste receptors are transmembrane protein network specialized in the transmission of information from special “sweet” molecules into the intracellular domain. These receptors can sense the taste of a range of molecules and transmit the information downstream to several acceptors, modulate cell specific functions and metabolism, and mediate cell-to-cell coupling through paracrine mechanism. Recent reports indicate that sweet taste receptors are widely distributed in the body and serves specific function relative to their localization. Due to their pleiotropic signaling properties and multisubstrate ligand affinity, sweet taste receptors are able to cooperatively bind multiple substances and mediate signaling by other receptors. Based on increasing evidence about the role of these receptors in the initiation and control of absorption and metabolism, and the pivotal role of metabolic (glucose regulation in the central nervous system functioning, we propose a possible implication of sweet taste receptor signaling in modulating cognitive functioning.

  5. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  6. Functionally biased signalling properties of 7TM receptors - opportunities for drug development for the ghrelin receptor

    DEFF Research Database (Denmark)

    Sivertsen, B; Holliday, N; Madsen, A N

    2013-01-01

    UNLABELLED: The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism...... and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several...... review, we have described how ligands and mutations in the 7TM receptor may bias the receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G-protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have...

  7. Emerging functions for neuropeptide Y5 receptors

    NARCIS (Netherlands)

    Bischoff, A.; Michel, M. C.

    1999-01-01

    The Y5 subtype of neuropeptide Y (NPY) receptors has raised considerable interest as a mediator of NPY-stimulated food intake, but with the advent of recent data, this hypothesis has come into question. Moreover, Y5 receptor-selective drugs might not be specific for food intake because additional

  8. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

    Science.gov (United States)

    Jørgensen, Kristine B; Krogh-Jensen, Karen; Pickering, Darryl S; Kanui, Titus I; Abelson, Klas S P

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

  9. NMDA receptor function during senescence: implication on cognitive performance

    Directory of Open Access Journals (Sweden)

    Ashok eKumar

    2015-12-01

    Full Text Available N-methyl-D-aspartate (NMDA receptors, a family of L-glutamate receptors, play an important role in learning and memory, and are critical for spatial memory. These receptors are tetrameric ion channels composed of a family of related subunits. One of the hallmarks of the aging human population is a decline in cognitive function; studies in the past couple of years have demonstrated deterioration in NMDA receptor subunit expression and function with advancing age. However, a direct relationship between impaired memory function and a decline in NMDA receptors is still ambiguous. Recent studies indicate a link between an age-associated NMDA receptor hypofunction and memory impairment and provide evidence that age-associated enhanced oxidative stress might be contributing to the alterations associated with senescence. However, clear evidence is still deficient in demonstrating the underlying mechanisms and a relationship between age-associated impaired cognitive faculties and NMDA receptor hypofunction. The current review intends to present an overview of the research findings regarding changes in expression of various NMDA receptor subunits and deficits in NMDA receptor function during senescence and its implication in age-associated impaired hippocampal-dependent memory function.

  10. Adenosine Receptor Heteromers and their Integrative Role in Striatal Function

    Directory of Open Access Journals (Sweden)

    Sergi Ferré

    2007-01-01

    Full Text Available By analyzing the functional role of adenosine receptor heteromers, we review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS. Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a “biochemical fingerprint” to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as “processors” of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the “local module” (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit, where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module.

  11. N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

    Science.gov (United States)

    Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

    2017-07-01

    N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

  12. Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

    Directory of Open Access Journals (Sweden)

    Fomina Alla F

    2008-09-01

    Full Text Available Abstract Background Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment. Results We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro. Conclusion This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

  13. Existence of 2m-1 Positive Solutions for Sturm-Liouville Boundary Value Problems with Linear Functional Boundary Conditions on the Half-Line

    Directory of Open Access Journals (Sweden)

    Yanmei Sun

    2012-01-01

    Full Text Available By using the Leggett-Williams fixed theorem, we establish the existence of multiple positive solutions for second-order nonhomogeneous Sturm-Liouville boundary value problems with linear functional boundary conditions. One explicit example with singularity is presented to demonstrate the application of our main results.

  14. Palmitoylation as a Functional Regulator of Neurotransmitter Receptors

    Directory of Open Access Journals (Sweden)

    Vladimir S. Naumenko

    2018-01-01

    Full Text Available The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs and ligand-gated ion channels (LICs. From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior.

  15. Melatonin membrane receptors in peripheral tissues: Distribution and functions

    Science.gov (United States)

    Slominski, Radomir M.; Reiter, Russel J.; Schlabritz-Loutsevitch, Natalia; Ostrom, Rennolds S.; Slominski, Andrzej T.

    2012-01-01

    Many of melatonin’s actions are mediated through interaction with the G-protein coupled membrane bound melatonin receptors type 1 and type 2 (MT1 and MT2, respectively) or, indirectly with nuclear orphan receptors from the RORα/RZR family. Melatonin also binds to the quinone reductase II enzyme, previously defined the MT3 receptor. Melatonin receptors are widely distributed in the body; herein we summarize their expression and actions in non-neural tissues. Several controversies still exist regarding, for example, whether melatonin binds the RORα/RZR family. Studies of the peripheral distribution of melatonin receptors are important since they are attractive targets for immunomodulation, regulation of endocrine, reproductive and cardiovascular functions, modulation of skin pigmentation, hair growth, cancerogenesis, and aging. Melatonin receptor agonists and antagonists have an exciting future since they could define multiple mechanisms by which melatonin modulates the complexity of such a wide variety of physiological and pathological processes. PMID:22245784

  16. Progesterone Receptor Scaffolding Function in Breast Cancer

    Science.gov (United States)

    2012-10-01

    leiomyoma cells in response to RU486 revealed little overlap [101]. PR-A and PR-B are most often co-expressed in the same tissues, and cells that... leiomyoma cells. PLoS One 7 (2012) e29021. [102]P.A. Mote, S. Bartow, N. Tran, C.L. Clarke, Loss of co-ordinate expression of progesterone receptors

  17. Steroid receptors and their ligands: Effects on male gamete functions

    International Nuclear Information System (INIS)

    Aquila, Saveria; De Amicis, Francesca

    2014-01-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  18. Steroid receptors and their ligands: Effects on male gamete functions

    Energy Technology Data Exchange (ETDEWEB)

    Aquila, Saveria; De Amicis, Francesca, E-mail: francesca.deamicis@unical.it

    2014-11-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  19. Hepatic macrophage complement receptor clearance function following injury.

    Science.gov (United States)

    Cuddy, B G; Loegering, D J; Blumenstock, F A; Shah, D M

    1986-03-01

    Previous work has demonstrated that in vivo hepatic macrophage complement receptor clearance function is depressed following thermal injury. The present study was carried out to determine if complement receptor function depression is associated with other states of depressed host defense. Hepatic complement receptor clearance function was determined from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM (EIgM) in rats. Receptor function was determined following cannulation of a carotid artery, laparotomy plus enterotomy, hemorrhagic shock, trauma, thermal injury, acute bacteremia, acute endotoxemia, and injection of erythrocyte stroma, gelatinized lipid emulsion, or colloidal carbon. Hepatic uptake of EIgM was depressed following each of these experimental interventions except arterial cannulation. This effect was shown not to be due to a decrease in hepatic blood flow or depletion of complement and was therefore due to a depression in hepatic macrophage complement receptor clearance function. Thus, impairment of hepatic macrophage complement receptor function is associated with several states of depressed host defense.

  20. Nature and regulation of the insulin receptor: structure and function

    International Nuclear Information System (INIS)

    Czech, M.P.

    1985-01-01

    Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner

  1. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

    1988-01-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

  2. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

    Science.gov (United States)

    Leonhardt, Julia; Villela, Daniel C; Teichmann, Anke; Münter, Lisa-Marie; Mayer, Magnus C; Mardahl, Maibritt; Kirsch, Sebastian; Namsolleck, Pawel; Lucht, Kristin; Benz, Verena; Alenina, Natalia; Daniell, Nicholas; Horiuchi, Masatsugu; Iwai, Masaru; Multhaup, Gerhard; Schülein, Ralf; Bader, Michael; Santos, Robson A; Unger, Thomas; Steckelings, Ulrike Muscha

    2017-06-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other. © 2017 American Heart Association, Inc.

  3. Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

    Science.gov (United States)

    Calebiro, Davide; Godbole, Amod

    2018-04-01

    G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Novel Functional Properties of Drosophila CNS Glutamate Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yan; Dharkar, Poorva; Han, Tae-Hee; Serpe, Mihaela; Lee, Chi-Hon; Mayer, Mark L.

    2016-12-01

    Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation.

  5. Molecular mechanisms of androgen receptor functions

    NARCIS (Netherlands)

    K. Steketee (Karine)

    2007-01-01

    textabstractThe androgens testosterone (T) and dihydrotestosterone (DHT) are steroid hormones, which are necessary for development and maintenance of the functions of the male sex organs, including the prostate. Androgens also play an important role in benign abnormalities of the prostate and in the

  6. Scavenger Receptor Structure and Function in Health and Disease

    Directory of Open Access Journals (Sweden)

    Izma Abdul Zani

    2015-05-01

    Full Text Available Scavenger receptors (SRs are a ‘superfamily’ of membrane-bound receptors that were initially thought to bind and internalize modified low-density lipoprotein (LDL, though it is currently known to bind to a variety of ligands including endogenous proteins and pathogens. New family of SRs and their properties have been identified in recent years, and have now been classified into 10 eukaryote families, defined as Classes A-J. These receptors are classified according to their sequences, although in each class they are further classified based in the variations of the sequence. Their ability to bind a range of ligands is reflected on the biological functions such as clearance of modified lipoproteins and pathogens. SR members regulate pathophysiological states including atherosclerosis, pathogen infections, immune surveillance, and cancer. Here, we review our current understanding of SR structure and function implicated in health and disease.

  7. Class I Cytokine Receptors: Structure and function in the Membrane

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard

    bilayer via structural characterizations of TMD representatives. To enable structural studies of these domains, an organic-extraction based strategy for efficient production of isotope-labeled TMDs with or without short intrinsically disordered regions was developed. This strategy successfully provided...... of these challenging domains. Supplemented by a review of the current collection of TMD structures from single-pass transmembrane receptors, the thesis as a whole provides important insights on the structure and function in the membrane as well as highlight the open questions to be addressed in the years to come.......Class I cytokine receptors are involved in important biological functions of both physiological and pathological nature in mammals. However, the molecular details of the cross-membrane signal transduction through these receptors remain obscure. One of the major reasons for this is the lack...

  8. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Tian, He; Naganathan, Saranga; Kazmi, Manija A

    2014-01-01

    Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

  9. TAAR1 Modulates Cortical Glutamate NMDA Receptor Function

    Science.gov (United States)

    Espinoza, Stefano; Lignani, Gabriele; Caffino, Lucia; Maggi, Silvia; Sukhanov, Ilya; Leo, Damiana; Mus, Liudmila; Emanuele, Marco; Ronzitti, Giuseppe; Harmeier, Anja; Medrihan, Lucian; Sotnikova, Tatyana D; Chieregatti, Evelina; Hoener, Marius C; Benfenati, Fabio; Tucci, Valter; Fumagalli, Fabio; Gainetdinov, Raul R

    2015-01-01

    Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor expressed in the mammalian brain and known to influence subcortical monoaminergic transmission. Monoamines, such as dopamine, also play an important role within the prefrontal cortex (PFC) circuitry, which is critically involved in high-o5rder cognitive processes. TAAR1-selective ligands have shown potential antipsychotic, antidepressant, and pro-cognitive effects in experimental animal models; however, it remains unclear whether TAAR1 can affect PFC-related processes and functions. In this study, we document a distinct pattern of expression of TAAR1 in the PFC, as well as altered subunit composition and deficient functionality of the glutamate N-methyl-D-aspartate (NMDA) receptors in the pyramidal neurons of layer V of PFC in mice lacking TAAR1. The dysregulated cortical glutamate transmission in TAAR1-KO mice was associated with aberrant behaviors in several tests, indicating a perseverative and impulsive phenotype of mutants. Conversely, pharmacological activation of TAAR1 with selective agonists reduced premature impulsive responses observed in the fixed-interval conditioning schedule in normal mice. Our study indicates that TAAR1 plays an important role in the modulation of NMDA receptor-mediated glutamate transmission in the PFC and related functions. Furthermore, these data suggest that the development of TAAR1-based drugs could provide a novel therapeutic approach for the treatment of disorders related to aberrant cortical functions. PMID:25749299

  10. Microsomal receptor for steroid hormones: functional implications for nuclear activity.

    Science.gov (United States)

    Muldoon, T G; Watson, G H; Evans, A C; Steinsapir, J

    1988-01-01

    Target tissues for steroid hormones are responsive by virtue of and to the extent of their content of functional intracellular receptors. Recent years have seen a shift in considerations of the cellular dynamics and distribution of these receptors, with current views favoring predominant intranuclear localization in the intact cell. This paper summarizes our analyses of the microsomal estrogen and androgen binding capability of rat uterine and ventral prostate tissue, respectively; these studies have revealed a set of high affinity sites that may act as a conduit for estrogen traversing the cell en route to the nucleus. These sites have many properties in common with cytosolic receptors, with the salient difference of a failure to activate to a more avid DNA-binding form under conditions which permit such activation of cytosolic receptors. The microsomal estrogen-binding proteins also have appreciable affinity for progesterone, another distinction from other known cellular estrogen receptor species. Various experimental approaches were employed to demonstrate that the microsomal receptors were not simply cytosol contaminants; the most convincing evidence is the recent successful separation of the cytosolic and microsomal forms by differential ammonium sulfate precipitation. Discrete subfractionation of subcellular components on successive sucrose gradients, with simultaneous assessments of binding capability and marker enzyme concentrations, indicates that the major portion of the binding is localized within the vesicles of the endoplasmic reticulum free of significant plasma membrane contamination. The microsomal receptors are readily solubilized by extraction with high- or low-salt-containing buffers or with steroid. The residual microsomes following such extraction have the characteristics of saturable acceptor sites for cytosolic estrogen-receptor complexes. The extent to which these sites will accept the cytosolic complexes is equal to the concentration of

  11. PTP1B regulates Eph receptor function and trafficking

    OpenAIRE

    Nievergall, Eva; Janes, Peter W.; Stegmayer, Carolin; Vail, Mary E.; Haj, Fawaz G.; Teng, Shyh Wei; Neel, Benjamin G.; Bastiaens, Philippe I.; Lackmann, Martin

    2010-01-01

    Eph receptors orchestrate cell positioning during normal and oncogenic development. Their function is spatially and temporally controlled by protein tyrosine phosphatases (PTPs), but the underlying mechanisms are unclear and the identity of most regulatory PTPs are unknown. We demonstrate here that PTP1B governs signaling and biological activity of EphA3. Changes in PTP1B expression significantly affect duration and amplitude of EphA3 phosphorylation and biological function, whereas confocal ...

  12. Identification of functional VEGF receptors on human platelets.

    Science.gov (United States)

    Selheim, Frode; Holmsen, Holm; Vassbotn, Flemming S

    2002-02-13

    Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

  13. The Efficacy of LY293558 in Blocking Seizures and Associated Morphological, and Behavioral Alterations Induced by Soman in Immature Male Rats and the Role of the M1 Muscarinic Acetylcholine Receptor in Organophosphate Induced Seizures

    Science.gov (United States)

    2015-01-30

    including seizures or status epilepticus (SE), and if left untreated results in long-term brain damage and neuropsychiatric symptoms or death. OPs...118 Abstract Exposure to nerve agents induces prolonged status epilepticus (SE), causing brain damage or death. Diazepam (DZP) is the presently...inhibition in the brain produces convulsive seizures and status epilepticus (SE), initiated by the excessive stimulation of cholinergic receptors. If

  14. NCS-1 associates with adenosine A2A receptors and modulates receptor function

    Directory of Open Access Journals (Sweden)

    Gemma eNavarro

    2012-04-01

    Full Text Available Modulation of G protein-coupled receptor (GPCR signalling by local changes in intracellular calcium concentration is an established function of Calmodulin which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with calmodulin targets with different functional outcome. In the present study we aimed to investigate if a target of calmodulin – the A2A adenosine receptor, is able to associate with two other neuronal calcium binding proteins, namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer and co-immunoprecipitation experiments we show the existence of A2A - NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signalling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signalling.

  15. Scavenger Receptor CD163 and Its Biological Functions

    Directory of Open Access Journals (Sweden)

    Gabriela Onofre

    2009-01-01

    Full Text Available CD163 is a member of scavenger receptor super family class B of the first subgroup. It is mapped to the region p13 on chromosome 12. Five different isoforms of CD163 have been described, which differ in the structure of their cytoplasmic domains and putative phosporylation sites. This scavenger receptor is selectively expressed on cells of monocytes and macrophages lineage exclusively. CD163 immunological function is essentially homeostatic. It also has other functions because participates in adhesion to endothelial cells, in tolerance induction and tissues regeneration. Other very important function of CD163 is the clearance of hemoglobin in its cell-free form and participation in anti-inflammation in its soluble form, exhibiting cytokine-like functions. We review the biological functions of CD163 which have been discovered until now. It seems apparent from this review that CD163 scavenger receptor can be used as biomarker in different diseases and as a valuable diagnostic parameter for prognosis of many diseases especially inflammatory disorders and sepsis.

  16. Expression and function of the human estrogen receptor in yeast

    International Nuclear Information System (INIS)

    White, J.H.; Metzger, D.; Chambon, P.

    1988-01-01

    Gene expression in eukaryotes is regulated at many levels. Moreover, there is increasing evidence that the basic control mechanisms of transcription initiation have been conserved across the range of eukaryotes from yeast to man. In vertebrates, the nuclear receptors, whose activity is dependent on the binding of specific ligands, stimulate transcription by interacting with specific cis-acting sequences and display all of the hallmarks of inducible enhancer factors. Alignment of their amino acid sequences indicates that they are composed of a series of conserved domains. The domain structure of the human estrogen receptor (hER) is typical of receptor proteins. Region C, containing two putative zinc fingers, comprises the DNA-binding domain responsible for specific recognition of estrogen response elements (ERE). Region E contains the hormone-binding domain and domain(s) responsible for transcription activation. A mutant of the hER, called HE15, which lacks the hormone-binding domain, binds DNA in vivo and in vitro but activates transcription only poorly in a constitutive manner in vivo in HeLa cells. A series of studies have demonstrated that the hormone- and DNA-binding domains of the nuclear receptors function independently. Chimeric proteins consisting of the DNA-binding domain of yeast GAL4 coupled to the hormone-binding domains of either the hER or glucocorticoid receptor element (GRE) will stimulate transcription in HeLa cells when bound to a UAS. Taken together, these results demonstrate that the hER and other nuclear receptors, as well as GAL4 and GCN4 proteins of yeast, consist of discrete and separable DNA-binding and transcription-activation functions. To investigate these striking parallels further, the authors have expressed the hER in the yeast Saccharomyces cerevisiae and have analyzed its hormone- and DNA-binding properties in vitro and its ability to stimulate transcription in vivo

  17. mGlu5 Receptor Functional Interactions and Addiction

    Directory of Open Access Journals (Sweden)

    Robyn eBrown

    2012-05-01

    Full Text Available The idea of ‘receptor mosaics’ suggests that proteins can form complex and dynamic networks, with respect to time and protein make up, which has the potential to make significant contributions to the diversity and specificity of GPCR signalling, particularly in neuropharmacology, where a few key receptors have been implicated in multiple neurological and psychiatric disorders such as addiction. Metabotropic glutamate type 5 receptors (mGlu5 have been shown to heterodimerise and form complexes with other GPCRs including adenosine A2A and dopamine D2 receptors. mGlu5-containing complexes are found in the striatum, a region of the brain known to be critical for mediating the rewarding and incentive motivational properties of drugs of abuse. Indeed, initial studies indicate a role for mGlu5-containing complexes in rewarding and conditioned effects of drugs, as well as drug-seeking behaviour. This is consistent with the substantial influence that mGlu5 complexes appear to have on striatal function, regulating both GABAergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents. Given their discrete localization, mGlu5-containing complexes represent a novel way in which to minimize the off-target effects and therefore provide us with an exciting therapeutic avenue for drug discovery efforts. Indeed, the therapeutic targeting of receptor mosaics in a tissue specific or temporal manner (for example, a sub-population of receptors in a ‘pathological state’ has the potential to dramatically reduce detrimental side effects that may otherwise impair vital brain function.

  18. Defensive and pathological functions of the gastrointestinal NK3 receptor.

    Science.gov (United States)

    Sanger, Gareth J; Tuladhar, Bishwa R; Bueno, Lionel; Furness, John B

    2006-10-01

    In general, normal gut functions are unaffected by selective NK(3) receptor antagonists such as talnetant (SB-223412), osanetant (SR 142901) or SB-235375. However, NK(3) receptors may mediate certain defensive or pathological intestinal processes. The precise mechanisms, by which this role is achieved, are not fully understood. In summary, intense stimulation of the intrinsic primary afferent neurones (IPANs) of the enteric nervous system is thought to release tachykinins from these neurones, to induce slow excitation (slow EPSPs) of connecting IPANs. This is hypothesised to cause hypersensitivity and disrupt intestinal motility, at least partly explaining why NK(3) receptor antagonism can reduce the level of disruption caused by supramaximal distension pressures in vitro. Tachykinin release from IPANs may also increase C-fibre sensitivity, directly or indirectly. Thus, NK(3) receptor antagonists can inhibit nociception associated with intestinal distension, in normal animals or after pre-sensitisation by restraint stress. Importantly, such inhibition has been found with SB-235375, a peripherally restricted antagonist. SB-235375 can also reduce a visceromotor response to brief colorectal distension without affecting similar responses to skin pinch, providing additional evidence for intestinal-specific activity. NK(3) receptor biology is, therefore, revealing a novel pathway by which disruptions in intestinal motility and nociception can be induced.

  19. Functional role of cannabinoid receptors in urinary bladder

    Directory of Open Access Journals (Sweden)

    Pradeep Tyagi

    2010-01-01

    Full Text Available Cannabinoids, the active components of Cannabis sativa (marijuana, and their derivatives produce a wide spectrum of central and peripheral effects, some of which may have clinical applications. The discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has attracted much attention to the general cannabinoid pharmacology. In recent years, studies on the functional role of cannabinoid receptors in bladder have been motivated by the therapeutic effects of cannabinoids on voiding dysfunction in multiple sclerosis patients. In this review, we shall summarize the literature on the expression of cannabinoid receptors in urinary bladder and the peripheral influence of locally and systemically administered cannabinoids in the bladder. The ongoing search for cannabinoid-based therapeutic strategies devoid of psychotropic effects can be complemented with local delivery into bladder by the intravesical route. A greater understanding of the role of the peripheral CB 1 and CB 2 receptor system in lower urinary tract is necessary to allow the development of new treatment for pelvic disorders.

  20. Bisphenol A affects androgen receptor function via multiple mechanisms.

    Science.gov (United States)

    Teng, Christina; Goodwin, Bonnie; Shockley, Keith; Xia, Menghang; Huang, Ruili; Norris, John; Merrick, B Alex; Jetten, Anton M; Austin, Christopher P; Tice, Raymond R

    2013-05-25

    Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10-100 nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1-2 μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2 μM), did not compete with R1881 for AR binding, when tested at 30 μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR. Published by Elsevier Ireland Ltd.

  1. Enhancing NMDA Receptor Function: Recent Progress on Allosteric Modulators

    Directory of Open Access Journals (Sweden)

    Lulu Yao

    2017-01-01

    Full Text Available The N-methyl-D-aspartate receptors (NMDARs are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer’s disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer’s disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs.

  2. Lactobacillus kefiranofaciens M1 isolated from milk kefir grains ameliorates experimental colitis in vitro and in vivo.

    Science.gov (United States)

    Chen, Y P; Hsiao, P J; Hong, W S; Dai, T Y; Chen, M J

    2012-01-01

    Lactobacillus kefiranofaciens M1, isolated from and identified in Taiwanese milk kefir grain, has demonstrated immune-modulating activity. In the present study, we further investigated the effects of Lb. kefiranofaciens M1 on intestinal epithelial cells in vitro and on dextran sodium sulfate (DSS)-induced colitis in vivo. The possible mechanisms regarding the cytokine products and intestinal epithelial barrier restoration as well as the putative receptor for the protective effects of Lb. kefiranofaciens M1 were investigated. In vitro results indicated that Lb. kefiranofaciens M1 could strengthen the epithelial barrier function in vitro by increasing the transepithelial electrical resistance (TEER) and significantly upregulated the level of the chemokine CCL-20 at both the apical and basolateral sites. The in vivo effects of Lb. kefiranofaciens M1 on the regulation of intestinal physiology indicate that this strain could ameliorate DSS-induced colitis with a significant attenuation of the bleeding score and colon length shortening. Production of proinflammatory cytokines was decreased and that of the antiinflammatory cytokine IL-10 was increased in the DSS-treated mice given Lb. kefiranofaciens M1. The putative receptor for the protective effects of Lb. kefiranofaciens M1 was toll-like receptor 2 (TLR2), which was involved in probiotic-induced cytokine production in vitro and in attenuation of the bleeding score and colon length shortening in vivo. In this study, the kefir lactobacillus Lb. kefiranofaciens M1 clearly demonstrated an anticolitis effect. Based on these results, Lb. kefiranofaciens M1 has the potential to be applied in fermented dairy products as an alternative therapy for intestinal disorders. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Integrating TRPV1 Receptor Function with Capsaicin Psychophysics

    Directory of Open Access Journals (Sweden)

    Gregory Smutzer

    2016-01-01

    Full Text Available Capsaicin is a naturally occurring vanilloid that causes a hot, pungent sensation in the human oral cavity. This trigeminal stimulus activates TRPV1 receptors and stimulates an influx of cations into sensory cells. TRPV1 receptors function as homotetramers that also respond to heat, proinflammatory substances, lipoxygenase products, resiniferatoxin, endocannabinoids, protons, and peptide toxins. Kinase-mediated phosphorylation of TRPV1 leads to increased sensitivity to both chemical and thermal stimuli. In contrast, desensitization occurs via a calcium-dependent mechanism that results in receptor dephosphorylation. Human psychophysical studies have shown that capsaicin is detected at nanomole amounts and causes desensitization in the oral cavity. Psychophysical studies further indicate that desensitization can be temporarily reversed in the oral cavity if stimulation with capsaicin is resumed at short interstimulus intervals. Pretreatment of lingual epithelium with capsaicin modulates the perception of several primary taste qualities. Also, sweet taste stimuli may decrease the intensity of capsaicin perception in the oral cavity. In addition, capsaicin perception and hedonic responses may be modified by diet. Psychophysical studies with capsaicin are consistent with recent findings that have identified TRPV1 channel modulation by phosphorylation and interactions with membrane inositol phospholipids. Future studies will further clarify the importance of capsaicin and its receptor in human health and nutrition.

  4. A novel M1 PAM VU0486846 exerts efficacy in cognition models without displaying agonist activity or cholinergic toxicity.

    Science.gov (United States)

    Rook, Jerri M; Bertron, Jeanette L; Cho, Hyekyung P; Garcia-Barrantes, Pedro M; Moran, Sean P; Maksymetz, James T; Nance, Kellie D; Dickerson, Jonathan W; Remke, Daniel H; Chang, Sichen; Harp, Joel; Blobaum, Anna L; Niswender, Colleen M; Jones, Carrie K; Stauffer, Shaun R; Conn, P Jeffrey; Lindsley, Craig W

    2018-04-27

    Selective activation of the M1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M1, leading to agonist actions in the prefrontal cortex (PFC) that impairs cognitive function, induces behavioral convulsions, and results in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M1-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427 and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [3H]-NMS binding at theorthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M1 PAM activity (EC50s > 100 nM) and pure-PAM activity in native tissues display robust pro-cognitive efficacy without AEs mediated by excessive activation of M1. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

  5. Molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Numa, S.; Fukuda, K.; Kubo, T.; Maeda, A.; Akiba, I.; Bujo, H.; Nakai, J.; Mishina, M.; Higashida, H.

    1988-01-01

    The muscarinic acetylcholine receptor (mAChR) mediates a variety of cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides, and modulation of potassium channels, through the action of guanine-nucleotide-binding regulatory proteins (G proteins). The question then arises as to whether multiple mAChR species exist that are responsible for the various biochemical and physiological effects. In fact, pharmacologically distinguishable forms of the mAChR occur in different tissues and have been provisionally classified into M 1 (I), M 2 cardiac (II), and M 2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. Here, the authors have made attempts to understand the molecular basis of the functional heterogeneity of the mAChR, using recombinant DNA technology

  6. Functionally Selective AT(1) Receptor Activation Reduces Ischemia Reperfusion Injury

    DEFF Research Database (Denmark)

    Hostrup, Anders; Christensen, Gitte Lund; Bentzen, Bo Hjort

    2012-01-01

    of the physiological functions of AngII. The AT(1)R mediates its effects through both G protein-dependent and independent signaling, which can be separated by functionally selective agonists. In the present study we investigate the effect of AngII and the ß-arrestin biased agonist [SII]AngII on ischemia......]AngII had a protective effect. Together these results demonstrate a cardioprotective effect of simultaneous blockade of G protein signaling and activation of G protein independent signaling through AT(1 )receptors....

  7. DMPD: Shaping of monocyte and macrophage function by adenosine receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17056121 Shaping of monocyte and macrophage function by adenosine receptors. Hasko ...tml) (.csml) Show Shaping of monocyte and macrophage function by adenosine receptors. PubmedID 17056121 Titl...e Shaping of monocyte and macrophage function by adenosine receptors. Authors Has

  8. Structure and function of the IFNγ receptor on human mononuclear phagocytes

    International Nuclear Information System (INIS)

    Schreiber, R.D.; Celada, A.

    1986-01-01

    Human mononuclear phagocytes bear a receptor that binds 125 I-IFNγ in a saturable, reversible and specific manner. The receptor consists minimally of a 70 kD polypeptide chain and its expression (5000/cell) and binding affinity (Ka=10 9 M -1 ) are unaffected by cellular activation or differentiation. The receptor's biological relevance was validated by correlating receptor occupancy with induction of a cellular response. 50% maximal induction of Fc receptors on U937 was effected by 0.8 nM IFNγ; the same concentration needed to half saturate U937 IFNγ receptors. Ligand-receptor interaction displayed species specificity but not cellular specificity. The receptors on U937 and human fibroblasts displayed identical ligand binding affinities (1.5-1.8 x 10 9 M -1 ). At 37 0 C, IFNγ bound to U937 in a biphasic manner. The high affinity binding component was due to ligand internalization since purified cell membranes and paraformaldehyde fixed cells displayed only the lower Ka and ligand internalization could be directly demonstrated. Using lysosomotropic amines, the internalized IFNγ-IFNγ receptor complex was tracked into an acid compartment where dissociation occurred. Free intracellular IFNγ was then degraded while free receptor entered an intracellular pool and eventually recycled back to the cell surface

  9. Influence of cochlear implantation on peripheral vestibular receptor function.

    Science.gov (United States)

    Krause, Eike; Louza, Julia P R; Wechtenbruch, Juliane; Gürkov, Robert

    2010-06-01

    The objectives of this study were 1) to assess the influence of a cochlear implantation on peripheral vestibular receptor function in the inner ear in the implant and in the nonimplant side, and 2) to analyze a possible correlation with resulting vertigo symptoms. Prospective clinical study. Cochlear implant center at tertiary referral hospital. A total of 32 patients, aged 15 to 83 years, undergoing cochlear implantation were assessed pre- and postoperatively for caloric horizontal semicircular canal response and vestibular-evoked myogenic potentials of the sacculus, and postoperatively for subjective vertigo symptoms. Patients with vertigo were compared with patients without symptoms with regard to the findings of the vestibular function tests. Cochlear implantation represents a significant risk factor for horizontal semicircular canal impairment (P 0.05). Cochlear implantation is a relevant risk factor for damage of peripheral vestibular receptor function. Therefore, preservation not only of residual hearing function but also of vestibular function should be aimed for, by using minimally invasive surgical techniques. Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.

  10. The pathophysiological functions mediated by D-1 dopamine receptors

    International Nuclear Information System (INIS)

    Goldstein, M.; Kuga, S.; Meller, E.; SHimizu, Y.

    1986-01-01

    This chapter describes some behavioral responses which might be mediated by D 1 and D 2 DA receptors, and the authors discuss their clinical relevance. It was of considerable interest to determine whether a selective D 1 DA antagonist, such as SCH 23390, will induce catalepsy and whether this behavior is mediated by D 1 , or by both D 1 and D 2 DA receptors. Rats were used in the experiments. The authors examined whether the addition of the S 2 antagonist ketanserin affects the displacement of 3 H-Spi by SCH 23390. Induction of self-mutilating biting (SMB) behavior in monkeys with unilateral ventromedial tegmental (VMT) lesions by DA agonists and its prevention by DA antagonists is examined. The authors also discuss the possible relationships between abnormal guanine nucleotide metabolism and dopaminergic neuronal function through the implications in LeschNyhan syndrome and in some mental disorders

  11. Functionality of the Paracoccidioides mating α-pheromone-receptor system.

    Directory of Open Access Journals (Sweden)

    Jéssica A Gomes-Rezende

    Full Text Available Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the α-pheromone (PBα gene, and the α- and a-pheromone receptor (PREB and PREA genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic α-pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to α-pheromone. In order to further evaluate the biological functionality of the pair α-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pbα pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active α-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBα in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides α-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.

  12. Influence of extracellular zinc on M1 microglial activation.

    Science.gov (United States)

    Higashi, Youichirou; Aratake, Takaaki; Shimizu, Shogo; Shimizu, Takahiro; Nakamura, Kumiko; Tsuda, Masayuki; Yawata, Toshio; Ueba, Tetuya; Saito, Motoaki

    2017-02-27

    Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30-60 μM ZnCl 2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia-reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.

  13. Central serotonergic and noradrenergic receptors in functional dyspepsia

    Institute of Scientific and Technical Information of China (English)

    S O'Mahony; TG Dinan; PW Keeling; ASB Chua

    2006-01-01

    Functional dyspepsia is a symptom complex characterised by upper abdominal discomfort or pain, early satiety,motor abnormalities, abdominal bloating and nausea in the absence of organic disease. The central nervous system plays an important role in the conducting and processing of visceral signals. Alterations in brain processing of pain, perception and affective responses may be key factors in the pathogenesis of functional dyspepsia. Central serotonergic and noradrenergic receptor systems are involved in the processing of motor,sensory and secretory activities of the gastrointestinal tract. Visceral hypersensitivity is currently regarded as the mechanism responsible for both motor alterations and abdominal pain in functional dyspepsia. Some studies suggest that there are alterations in central serotonergic and noradrenergic systems which may partially explain some of the symptoms of functional dyspepsia. Alterations in the autonomic nervous system may be implicated in the motor abnormalities and increases in visceral sensitivity in these patients.Noradrenaline is the main neurotransmitter in the sympathetic nervous system and again alterations in the functioning of this system may lead to changes in motor function. Functional dyspepsia causes considerable burden on the patient and society. The pathophysiology of functional dyspepsia is not fully understood but alterations in central processing by the serotonergic and noradrenergic systems may provide plausible explanations for at least some of the symptoms and offer possible treatment targets for the future.

  14. Transceptors as a functional link of transporters and receptors

    Directory of Open Access Journals (Sweden)

    George Diallinas

    2017-03-01

    Full Text Available Cells need to communicate with their environment in order to obtain nutrients, grow, divide and respond to signals related to adaptation in changing physiological conditions or stress. A very basic question in biology is how cells, especially of those organisms living in rapidly changing habitats, sense their environment. Apparently, this question is of particular importance to all free-living microorganisms. The critical role of receptors, transporters and channels, transmembrane proteins located in the plasma membrane of all types of cells, in signaling environmental changes is well established. A relative newcomer in environment sensing are the so called transceptors, membrane proteins that possess both solute transport and receptor-like signaling activities. Now, the transceptor concept is further enlarged to include micronutrient sensing via the iron and zinc high-affinity transporters of Saccharomyces cerevisiae. Interestingly, what seems to underline the transport and/or sensing function of receptors, transporters and transceptors is ligand-induced conformational alterations recognized by downstream intracellular effectors.

  15. Unique interaction pattern for a functionally biased ghrelin receptor agonist

    DEFF Research Database (Denmark)

    Sivertsen, Bjørn Behrens; Lang, Manja; Frimurer, Thomas M.

    2011-01-01

    Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers...... connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained w......Fw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Ga(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Ga(12...

  16. Megalin functions as an endocytic sonic hedgehog receptor.

    Science.gov (United States)

    McCarthy, Robert A; Barth, Jeremy L; Chintalapudi, Mastan R; Knaak, Christian; Argraves, W Scott

    2002-07-12

    Embryos deficient in the morphogen Sonic hedgehog (Shh) or the endocytic receptor megalin exhibit common neurodevelopmental abnormalities. Therefore, we have investigated the possibility that a functional relationship exists between the two proteins. During embryonic development, megalin was found to be expressed along the apical surfaces of neuroepithelial cells and was coexpressed with Shh in the ventral floor plate of the neural tube. Using enzyme-linked immunosorbent assay, homologous ligand displacement, and surface plasmon resonance techniques, it was found that the amino-terminal fragment of Shh (N-Shh) bound to megalin with high affinity. Megalin-expressing cells internalized N-Shh through a mechanism that was inhibited by antagonists of megalin, viz. anti-receptor-associated protein and anti-megalin antibodies. Heparin also inhibited N-Shh endocytosis, implicating proteoglycans in the internalization process, as has been described for other megalin ligands. Use of chloroquine to inhibit lysosomal proteinase activity showed that N-Shh endocytosed via megalin was not efficiently targeted to the lysosomes for degradation. The ability of megalin-internalized N-Shh to bypass lysosomes may relate to the finding that the interaction between N-Shh and megalin was resistant to dissociation with low pH. Together, these findings show that megalin is an efficient endocytic receptor for N-Shh. Furthermore, they implicate megalin as a new regulatory component of the Shh signaling pathway.

  17. Cardiac microvascular endothelial cells express a functional Ca+ -sensing receptor.

    Science.gov (United States)

    Berra Romani, Roberto; Raqeeb, Abdul; Laforenza, Umberto; Scaffino, Manuela Federica; Moccia, Francesco; Avelino-Cruz, Josè Everardo; Oldani, Amanda; Coltrini, Daniela; Milesi, Veronica; Taglietti, Vanni; Tanzi, Franco

    2009-01-01

    The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores. Copyright 2008 S. Karger AG, Basel.

  18. Selective androgen receptor modulators as function promoting therapies.

    Science.gov (United States)

    Bhasin, Shalender; Jasuja, Ravi

    2009-05-01

    The past decade has witnessed an unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Although steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5alpha-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with androgen receptor (AR) contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand-binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis.

  19. The First Fifteen Years of Steroid Receptor Research in Zebrafish; Characterization and Functional Analysis of the Receptors

    Directory of Open Access Journals (Sweden)

    Marcel J. M. Schaaf

    2017-07-01

    Full Text Available Steroid hormones regulate a wide range of processes in our body, and their effects are mediated by steroid receptors. In addition to their physiological role, these receptors mediate the effects of endocrine disrupting chemicals (EDCs and are widely used targets for dugs involved in the treatment of numerous diseases, ranging from cancer to inflammatory disorders. Over the last fifteen years, the zebrafish has increasingly been used as an animal model in steroid receptor research. Orthologues of all human steroid receptor genes appear to be present in zebrafish. All zebrafish steroid receptors have been characterized in detail, and their expression patterns have been analyzed. Functional studies have been performed using morpholino knockdown of receptor expression and zebrafish lines carrying mutations in one of their steroid receptor genes. To investigate the activity of the receptors in vivo, specific zebrafish reporter lines have been developed, and transcriptomic studies have been carried out to identify biomarkers for steroid receptor action. In this review, an overview of research on steroid receptors in zebrafish is presented, and it is concluded that further exploitation of the possibilities of the zebrafish model system will contribute significantly to the advancement of steroid receptor research in the next decade.

  20. The CCK(-like) receptor in the animal kingdom: functions, evolution and structures.

    Science.gov (United States)

    Staljanssens, Dorien; Azari, Elnaz Karimian; Christiaens, Olivier; Beaufays, Jérôme; Lins, Laurence; Van Camp, John; Smagghe, Guy

    2011-03-01

    In this review, the cholecystokinin (CCK)(-like) receptors throughout the animal kingdom are compared on the level of physiological functions, evolutionary basis and molecular structure. In vertebrates, the CCK receptor is an important member of the G-protein coupled receptors as it is involved in the regulation of many physiological functions like satiety, gastrointestinal motility, gastric acid secretion, gall bladder contraction, pancreatic secretion, panic, anxiety and memory and learning processes. A homolog for this receptor is also found in nematodes and arthropods, called CK receptor and sulfakinin (SK) receptor, respectively. These receptors seem to have evolved from a common ancestor which is probably still closely related to the nematode CK receptor. The SK receptor is more closely related to the CCK receptor and seems to have similar functions. A molecular 3D-model for the CCK receptor type 1 has been built together with the docking of the natural ligands for the CCK and SK receptors in the CCK receptor type 1. These molecular models can help to study ligand-receptor interactions, that can in turn be useful in the development of new CCK(-like) receptor agonists and antagonists with beneficial health effects in humans or potential for pest control. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  2. Functional interaction between Lypd6 and nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with n...... brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit n......AChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our...

  3. The vitamin d receptor and T cell function

    DEFF Research Database (Denmark)

    Kongsbak, Martin; Levring, Trine B; Geisler, Carsten

    2013-01-01

    The vitamin D receptor (VDR) is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH)2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH)2D3-VDR signaling on immune...... function has been the focus of many recent studies as a link between 1,25(OH)2D3 and susceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR...... ultimately to increase their chance of survival. Immune modulatory therapies that enhance VDR expression and activity are therefore considered in the clinic today to a greater extent. As T cells are of great importance for both protective immunity and development of inflammatory diseases a variety of studies...

  4. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

  5. The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    Science.gov (United States)

    Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

    2009-10-01

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

  6. Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways.

    Science.gov (United States)

    Tomàs, Josep; Garcia, Neus; Lanuza, Maria A; Santafé, Manel M; Tomàs, Marta; Nadal, Laura; Hurtado, Erica; Simó-Ollé, Anna; Cilleros-Mañé, Víctor; Just-Borràs, Laia

    2018-01-01

    In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR) in the mammalian neuromuscular junction (NMJ). Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells) cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally). These observations underlie the relevance of AR in the NMJ function.

  7. Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways

    Directory of Open Access Journals (Sweden)

    Josep Tomàs

    2018-04-01

    Full Text Available In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR in the mammalian neuromuscular junction (NMJ. Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally. These observations underlie the relevance of AR in the NMJ function.

  8. Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

    OpenAIRE

    Levoye, Angélique; Dam, Julie; Ayoub, Mohammed A; Guillaume, Jean-Luc; Jockers, Ralf

    2006-01-01

    G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observ...

  9. Rapid resensitization of purinergic receptor function in human platelets.

    Science.gov (United States)

    Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

    2008-08-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

  10. Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus.

    Directory of Open Access Journals (Sweden)

    Weiwei Lei

    Full Text Available Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of Tas2r (bitter receptor genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in

  11. Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus).

    Science.gov (United States)

    Lei, Weiwei; Ravoninjohary, Aurore; Li, Xia; Margolskee, Robert F; Reed, Danielle R; Beauchamp, Gary K; Jiang, Peihua

    2015-01-01

    Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of Tas2r (bitter receptor) genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus) should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in understanding the forces

  12. Lamin B Receptor: Interplay between Structure, Function and Localization

    Directory of Open Access Journals (Sweden)

    Eleni Nikolakaki

    2017-08-01

    Full Text Available Lamin B receptor (LBR is an integral protein of the inner nuclear membrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophobic segments that span the membrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have been attributed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contributing to the shape of interphase nuclear architecture, while its transmembrane domains exhibit sterol reductase activity. Mutations within the transmembrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and Greenberg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic behavior have not yet been fully unraveled. Here, we provide an overview of the current knowledge of the interplay between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR.

  13. The vitamin D receptor and T cell function

    Directory of Open Access Journals (Sweden)

    Martin eKongsbak

    2013-06-01

    Full Text Available The vitamin D receptor (VDR is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH2D3-VDR signaling on immune function has been the focus of many recent studies as a link between 1,25(OH2D3 and sus-ceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR ultimately to increase their chance of survival. Immune modulatory therapies that enhance VDR expression and activity are therefore considered in the clinic today to a greater extent. As T cells are of great importance for both protective immunity and development of inflammatory diseases a variety of studies have been engaged investigating the impact of VDR ex-pression in T cells and found that VDR expression and activity plays an important role in both T cell development, differentiation and effector function. In this review we will analyze current know-ledge of VDR regulation and function in T cells and discuss its importance for immune activity.

  14. Do unliganded thyroid hormone receptors have physiological functions?

    Science.gov (United States)

    Chassande, O

    2003-08-01

    Thyroid hormone (TH) is required for the development of vertebrates and exerts numerous homeostatic functions in adults. TH acts through nuclear receptors which control the transcription of target genes. Unliganded and liganded thyroid hormone receptors (TRs) have been shown to exert opposite effects on the transcription of target genes in vitro. However, the occurance of an aporeceptor activity in vivo and its potential physiological significance has not been clearly addressed. Several data generated using experimental hypothyroidism and thyrotoxicosis in wild type and TR knockout mice support the notion that apoTRs have an intrinsic activity in several tIssues. ApoTRs, and in particular TRalpha1, are predominant during the early stages of vertebrate development and must be turned into holoTRs for post-natal development to proceed normally. However, the absence of striking alterations of embryonic and fetal development in mice devoid of TRs indicates that apoTRs do not play a fundamental role. During development, as well as in adults, apoTRs rather appears as a system which increases the range of transcriptional responses to moderate variations of T3.

  15. Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

    Directory of Open Access Journals (Sweden)

    Donghyun Kim

    2017-07-01

    Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

  16. Structure and function of the human megalin receptor

    DEFF Research Database (Denmark)

    Dagil, Robert

    . The extracellular domain of megalin consists of several modular domains, of which the most abundant are the ligand binding complement type (CR) domains, that are divided into four clusters separated by YWTD -propeller domains. The broad ligand binding profile has associated megalin with the unwanted cellular uptake...... of aminoglycosides during antibacterial treatment, which can lead to nephro- and ototoxic side-effects. This thesis presents new insights into the structure-function relation of the megalin receptor. The interaction between megalin and several natural protein ligands as well as the aminoglycoside gentamicin...... were involved in binding, which utilizes the commonly found ligand binding motif. The details of the atomic resolutionmodel will aid the future design of effective megalin antagonists, however, since the common ligand binding motif of CR domains is used to bind gentamicin this may not be a trivial task...

  17. Molecular determinants of odorant receptor function in insects

    Indian Academy of Sciences (India)

    2014-07-20

    Jul 20, 2014 ... other host-odor responsive receptors from vector insect spe- cies would .... those that mediate host-seeking behaviour in insect disease vectors and ... receptors are transmitted and processed via olfactory circuits. (Vosshall ...

  18. Functional properties of Virus-Encoded and Virus-Regulated 7TM Receptors

    DEFF Research Database (Denmark)

    Spiess, Katja; Rosenkilde, Mette Marie

    2014-01-01

    During co-evolution with their hosts, viruses have developed several survival strategies that involve exploitation of 7TM receptors. These include virus-encoded 7TM receptors and ligands and viral regulation of endogenous receptors. Many functional properties have been ascribed to virus-exploited...

  19. Functional evolution of the vitamin D and pregnane X receptors

    Directory of Open Access Journals (Sweden)

    Ou Junhai

    2007-11-01

    Full Text Available Abstract Background The vitamin D receptor (VDR and pregnane X receptor (PXR are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt. VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus, zebrafish (Danio rerio, and African clawed frog (Xenopus laevis. Results Human, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6β-hydroxylation activity (a measure of cytochrome P450 3A activity in other species and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5, following injection of the major zebrafish bile salt, 5α-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene. Conclusion Our results show tight conservation of ligand

  20. Selective Androgen Receptor Modulators (SARMs) as Function Promoting Therapies

    Science.gov (United States)

    Bhasin, Shalender; Jasuja, Ravi

    2010-01-01

    Purpose of review The last decade has witnessed unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Recent Findings While steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5α-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with AR contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. Summary SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis. PMID:19357508

  1. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

    DEFF Research Database (Denmark)

    Leonhardt, Julia; Villela, Daniel C.; Teichmann, Anke

    2017-01-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may in...

  2. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct...... receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known...... revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor...

  3. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    International Nuclear Information System (INIS)

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J.

    1990-01-01

    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed

  4. Serotonin and brain function: a tale of two receptors.

    Science.gov (United States)

    Carhart-Harris, R L; Nutt, D J

    2017-09-01

    Previous attempts to identify a unified theory of brain serotonin function have largely failed to achieve consensus. In this present synthesis, we integrate previous perspectives with new and older data to create a novel bipartite model centred on the view that serotonin neurotransmission enhances two distinct adaptive responses to adversity, mediated in large part by its two most prevalent and researched brain receptors: the 5-HT1A and 5-HT2A receptors. We propose that passive coping (i.e. tolerating a source of stress) is mediated by postsynaptic 5-HT1AR signalling and characterised by stress moderation. Conversely, we argue that active coping (i.e. actively addressing a source of stress) is mediated by 5-HT2AR signalling and characterised by enhanced plasticity (defined as capacity for change). We propose that 5-HT1AR-mediated stress moderation may be the brain's default response to adversity but that an improved ability to change one's situation and/or relationship to it via 5-HT2AR-mediated plasticity may also be important - and increasingly so as the level of adversity reaches a critical point. We propose that the 5-HT1AR pathway is enhanced by conventional 5-HT reuptake blocking antidepressants such as the selective serotonin reuptake inhibitors (SSRIs), whereas the 5-HT2AR pathway is enhanced by 5-HT2AR-agonist psychedelics. This bipartite model purports to explain how different drugs (SSRIs and psychedelics) that modulate the serotonergic system in different ways, can achieve complementary adaptive and potentially therapeutic outcomes.

  5. Genetic, functional and molecular features of glucocorticoid receptor binding.

    Directory of Open Access Journals (Sweden)

    Francesca Luca

    Full Text Available Glucocorticoids (GCs are key mediators of stress response and are widely used as pharmacological agents to treat immune diseases, such as asthma and inflammatory bowel disease, and certain types of cancer. GCs act mainly by activating the GC receptor (GR, which interacts with other transcription factors to regulate gene expression. Here, we combined different functional genomics approaches to gain molecular insights into the mechanisms of action of GC. By profiling the transcriptional response to GC over time in 4 Yoruba (YRI and 4 Tuscans (TSI lymphoblastoid cell lines (LCLs, we suggest that the transcriptional response to GC is variable not only in time, but also in direction (positive or negative depending on the presence of specific interacting transcription factors. Accordingly, when we performed ChIP-seq for GR and NF-κB in two YRI LCLs treated with GC or with vehicle control, we observed that features of GR binding sites differ for up- and down-regulated genes. Finally, we show that eQTLs that affect expression patterns only in the presence of GC are 1.9-fold more likely to occur in GR binding sites, compared to eQTLs that affect expression only in its absence. Our results indicate that genetic variation at GR and interacting transcription factors binding sites influences variability in gene expression, and attest to the power of combining different functional genomic approaches.

  6. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Directory of Open Access Journals (Sweden)

    Gerd Krause

    2017-04-01

    Full Text Available The thyroid-stimulating hormone receptor (TSHR is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs. TSHR and its endogenous ligand thyrotropin (TSH are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016 concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other

  7. Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  8. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  9. m=1” coatings

    DEFF Research Database (Denmark)

    Cooper-Jensen, C.; Klinkby, Esben Bryndt; Beaucour, J.

    For neutrons the critical angle of Ni is defined as m=1. If one needs a coating with reflectivity above m=1, people have traditionally used Ni58 or Ni-based multilayers. The reason to use Ni is the high neutron scattering density and the fact that it is easy to coat Ni using magnetron sputtering....... For a neutron guide the cost of shielding around the guide is a substantial part of the total cost of the guide. We are therefore looking at other materials than Ni for m=1 coatings. Both Be and diamond have the same or higher neutron scattering density than Ni, and have a much smaller absorption cross section....... Because of the lower absorption cross section, and because of fewer emitted gamma ray photons when a neutron is absorbed, these coatings are producing much less gamma radiation and therefore reduce the shielding costs. Be is frequently used in a wide range of science and technology applications. The only...

  10. Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis

    DEFF Research Database (Denmark)

    Husted, L B; Harsløf, T; Stenkjær, L

    2013-01-01

    variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse......UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three...... of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted...

  11. The Neuroendocrine Functions of the Parathyroid Hormone 2 Receptor

    Directory of Open Access Journals (Sweden)

    Arpad eDobolyi

    2012-10-01

    Full Text Available The G-protein coupled parathyroid hormone 2 receptor (PTH2R is concentrated in endocrine and limbic regions in the forebrain. Its endogenous ligand,tuberoinfundibular peptide of 39 residues (TIP39, is synthesized in only 2 brain regions, within the posterior thalamus and the lateral pons. TIP39-expressing neurons have a widespread projection pattern, which matches the PTH2R distribution in the brain. Neuroendocrine centers including the preoptic area, the periventricular, paraventricular, and arcuate nuclei contain the highest density of PTH2R-positive networks. The administration of TIP39 and an antagonist of the PTH2R as well as the investigation of mice that lack functional TIP39 and PTH2R revealed the involvement of the PTH2R in a variety of neural and neuroendocrine functions. TIP39 acting via the PTH2R modulates several aspects of the stress response. It evokes corticosterone release by activating corticotropin-releasing hormone-containing neurons in the hypothalamic paraventricular nucleus. Block of TIP39 signaling elevates the anxiety state of animals and their fear response, and increases stress-induced analgesia. TIP39 has also been suggested to affect the release of additional pituitary hormones including arginine vasopressin and growth hormone. A role of the TIP39-PTH2R system in thermoregulation was also identified. TIP39 may play a role in maintaining body temperature in a cold environment via descending excitatory pathways from the preoptic area. Anatomical and functional studies also implicated the TIP39-PTH2R system in nociceptive information processing. Finally, TIP39 induced in postpartum dams may play a role in the release of prolactin during lactation. Potential mechanisms leading to the activation of TIP39 neurons and how they influence the neuroendocrine system are also described. The unique TIP39-PTH2R neuromodulator system provides the possibility for developing drugs with a novel mechanism of action to control

  12. Functional expression of the 5-HT1c receptor in neuronal and nonneuronal cells

    International Nuclear Information System (INIS)

    Julius, D.; MacDermott, A.B.; Jessel, T.M.; Huang, K.; Molineaux, S.; Schieren, I.; Axel, R.

    1988-01-01

    The isolation of the genes encoding the multiple serotonin receptor subtypes and the ability to express these receptors in new cellular environments will help to elucidate the molecular mechanisms of action of serotonin in the mammalian brain. The cloning of most neurotransmitter receptors has required the purification of receptor, the determination of partial protein sequence, and the synthesis of oligonucleotide probes with which to obtain cDNA or genomic clones. However, the serotonin receptors have not been purified and antibodies have not been generated. The authors therefore designed a cDNA expression system that permits the identification of functional cDNA clones encoding serotonin receptors in the absence of protein sequence information. They have combined cloning in RNA expression vectors with an electrophysiological assay in oocytes to isolate a functional cDNA clone encoding the entire 5-HT 1c receptor. The sequence of this clone reveals that the 5-HT 1c receptor belongs to a family of G-protein-coupled receptors that are thought to traverse the membrane seven times. Mouse fibroblasts transformed with this clone bind serotonergic ligands and respond to serotonin with an elevation in intracellular calcium. Moreover, in situ hybridization and Northern blot analysis indicate that the 5-HT 1c receptor mRNA is expressed in a wide variety of neurons in the rat central nervous system, suggesting that this receptor plays a prominent role in neuronal function

  13. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  14. Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

    NARCIS (Netherlands)

    Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

    2001-01-01

    Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

  15. Predicting Trainability of M1 Crewmen

    Science.gov (United States)

    1982-10-01

    Load Main Gun Clear Main Gun LOAD/UNLOAD M250 GRENADE LAUNCHER ON M1 TANK* Load Grenade Launcher Unload Grenade Launcher PREPARE GUNNER’S STATION...Clear Main Gun LOAD/UNLOAD M250 GRENADE LAUNCHER ON Ml TANK* Load Grenade Launcher Unload Grenade Lauacher PREPARE GUNNER’S STATION FOR OPERATION ON Ml

  16. Modern approaches to the design of memory and cognitive function stimulants based on AMPA receptor ligands

    International Nuclear Information System (INIS)

    Grigoriev, V V; Proshin, A N; Kinzirsky, A S; Bachurin, Sergey O

    2009-01-01

    Data on the structure and properties of compounds acting on AMPA receptors, the key subtype of ionotropic glutamate receptors of the mammalian central nervous system, are analyzed. Data on the role of these receptors in provision of memory and cognitive function formation and impairment processes are presented. The attention is focused on the modern views on the mechanisms of AMPA receptor desensitization and deactivation and action of substances affecting these processes. The structures of key positive modulators of AMPA receptors are given. The problems of application of these substances as therapeutic means for preventing and treating neurodegenerative and psychoneurological diseases are discussed. Bibliography - 121 references.

  17. Modern approaches to the design of memory and cognitive function stimulants based on AMPA receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriev, V V; Proshin, A N; Kinzirsky, A S; Bachurin, Sergey O [Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolovka, Moscow Region (Russian Federation)

    2009-05-31

    Data on the structure and properties of compounds acting on AMPA receptors, the key subtype of ionotropic glutamate receptors of the mammalian central nervous system, are analyzed. Data on the role of these receptors in provision of memory and cognitive function formation and impairment processes are presented. The attention is focused on the modern views on the mechanisms of AMPA receptor desensitization and deactivation and action of substances affecting these processes. The structures of key positive modulators of AMPA receptors are given. The problems of application of these substances as therapeutic means for preventing and treating neurodegenerative and psychoneurological diseases are discussed. Bibliography - 121 references.

  18. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  19. Modern approaches to the design of memory and cognitive function stimulants based on AMPA receptor ligands

    Science.gov (United States)

    Grigoriev, V. V.; Proshin, A. N.; Kinzirsky, A. S.; Bachurin, Sergey O.

    2009-05-01

    Data on the structure and properties of compounds acting on AMPA receptors, the key subtype of ionotropic glutamate receptors of the mammalian central nervous system, are analyzed. Data on the role of these receptors in provision of memory and cognitive function formation and impairment processes are presented. The attention is focused on the modern views on the mechanisms of AMPA receptor desensitization and deactivation and action of substances affecting these processes. The structures of key positive modulators of AMPA receptors are given. The problems of application of these substances as therapeutic means for preventing and treating neurodegenerative and psychoneurological diseases are discussed. Bibliography — 121 references.

  20. Minoxidil may suppress androgen receptor-related functions.

    Science.gov (United States)

    Hsu, Cheng-Lung; Liu, Jai-Shin; Lin, An-Chi; Yang, Chih-Hsun; Chung, Wen-Hung; Wu, Wen-Guey

    2014-04-30

    Although minoxidil has been used for more than two decades to treat androgenetic alopecia (AGA), an androgen-androgen receptor (AR) pathway-dominant disease, its precise mechanism of action remains elusive. We hypothesized that minoxidil may influence the AR or its downstream signaling. These tests revealed that minoxidil suppressed AR-related functions, decreasing AR transcriptional activity in reporter assays, reducing expression of AR targets at the protein level, and suppressing AR-positive LNCaP cell growth. Dissecting the underlying mechanisms, we found that minoxidil interfered with AR-peptide, AR-coregulator, and AR N/C-terminal interactions, as well as AR protein stability. Furthermore, a crystallographic analysis using the AR ligand-binding domain (LBD) revealed direct binding of minoxidil to the AR in a minoxidil-AR-LBD co-crystal model, and surface plasmon resonance assays demonstrated that minoxidil directly bound the AR with a K(d) value of 2.6 µM. Minoxidil also suppressed AR-responsive reporter activity and decreased AR protein stability in human hair dermal papilla cells. The current findings provide evidence that minoxidil could be used to treat both cancer and age-related disease, and open a new avenue for applications of minoxidil in treating androgen-AR pathway-related diseases.

  1. The meth brain: methamphetamines alter brain functions via NMDA receptors

    Czech Academy of Sciences Publication Activity Database

    Proft, Juliane; Weiss, Norbert

    2015-01-01

    Roč. 34, č. 1 (2015), s. 1-3 ISSN 0231-5882 R&D Projects: GA ČR GA15-13556S Institutional support: RVO:61388963 Keywords : ion channel * methamphetamine * piriform cortex * NMDA receptor * AMPA receptor Subject RIV: CE - Biochemistry Impact factor: 0.892, year: 2015

  2. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...... in normal human intestinal epithelia and could play a role in cholera....

  3. Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hansen, M.K.; Tams, J.W.; Fahrenkrug, Jan

    1999-01-01

    G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide......G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide...

  4. Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

    Science.gov (United States)

    Gelband, C H; Sumners, C; Lu, D; Raizada, M K

    1998-02-27

    The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

  5. EphA2 is a functional receptor for the growth factor progranulin.

    Science.gov (United States)

    Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

    2016-12-05

    Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

  6. Functional relevance of neurotransmitter receptor heteromers in the central nervous system.

    Science.gov (United States)

    Ferré, Sergi; Ciruela, Francisco; Woods, Amina S; Lluis, Carme; Franco, Rafael

    2007-09-01

    The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. Heteromerization of neurotransmitter receptors produces functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Neurotransmitter receptor heteromers can function as processors of computations that modulate cell signaling. Thus, the quantitative or qualitative aspects of the signaling generated by stimulation of any of the individual receptor units in the heteromer are different from those obtained during coactivation. Furthermore, recent studies demonstrate that some neurotransmitter receptor heteromers can exert an effect as processors of computations that directly modulate both pre- and postsynaptic neurotransmission. This is illustrated by the analysis of striatal receptor heteromers that control striatal glutamatergic neurotransmission.

  7. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Science.gov (United States)

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-09-01

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Structure-function relationships for the interleukin 2 receptor system

    Directory of Open Access Journals (Sweden)

    Richard J. Robb

    1987-01-01

    Full Text Available Receptors for interleukin 2 (IL-2 esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta] chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

  9. Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking

    DEFF Research Database (Denmark)

    Roed, Sarah Noerklit; Nøhr, Anne Cathrine; Wismann, Pernille

    2015-01-01

    The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have......) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue...... coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression...

  10. Angiotensin II Type 2 Receptor and Receptor Mas Are Colocalized and Functionally Interdependent in Obese Zucker Rat Kidney

    DEFF Research Database (Denmark)

    Patel, Sanket N; Ali, Quaisar; Samuel, Preethi

    2017-01-01

    The actions of angiotensin II type 2 receptor (AT2R) and the receptor Mas (MasR) are complex but show similar pronatriuretic function; particularly, AT2R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of some reports suggesting a potential positive...... interaction between these receptors, we tested hypothesis that renal AT2R and MasR physically interact and are interdependent to stimulate cell signaling and promote natriuresis in obese rats. We found that infusion of AT2R agonist C21 in obese Zucker rats (OZR) increased urine flow and urinary Na excretion...... coimmunoprecipitated with MasR in cortical homogenate of OZR. Immunoblotting of cortical homogenate cross-linked with zero-length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline revealed a shift of AT2R and MasR bands upward with overlapping migration for their complexes which were sensitive...

  11. [GPCRs heterodimerization: a new way towards the discovery of function for the orphan receptors?].

    Science.gov (United States)

    Levoye, Angélique; Jockers, Ralf

    2007-01-01

    G protein-coupled receptors (GPCRs), also called seven transmembrane domain (7TM) proteins, represent the largest family of cell surface receptors. GPCRs control a variety of physiological processes, are involved in multiple diseases and are major drug targets. Despite a vast effort of academic and industrial research, more than one hundred receptors remain orphans. These orphan GPCRs offer a great potential for drug discovery, as almost 60% of currently prescribed drugs target GPCRs. Deorphenization strategies have concentrated mainly on the identification of the natural ligands of these proteins. Recent advances have shown that orphan GPCRs, similar to orphan nuclear receptors, can regulate the function of non-orphan receptors by heterodimerization. These findings not only help to better understand the extraordinary diversity of GPCRs, but also open new perspectives for the identification of the function of these orphan receptors that hold great therapeutic potential.

  12. Molecular Mechanisms of β2-Adrenergic Receptor Function and Regulation

    OpenAIRE

    McGraw, Dennis W.; Liggett, Stephen B.

    2005-01-01

    It is now clear that the β2-adrenergic receptor continuously oscillates between various conformations in the basal state, and that agonists act to stabilize one or more conformations. It is conceivable that synthetic agonists might be engineered to preferentially confine the receptor to certain conformations deemed clinically important while having a less stabilizing effect on unwanted conformations. In addition, studies of genetically engineered mice have revealed previously unrecognized cro...

  13. AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

    Science.gov (United States)

    Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

    2017-01-01

    The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2013-04-01

    Full Text Available Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer.

  15. GABA_A receptor function is regulated by lipid bilayer elasticity

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Werge, Thomas; Berthelsen, Camilla

    2006-01-01

    ( s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABAA receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown...... reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABAA receptor function is regulated by lipid bilayer elasticity....... PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer....

  16. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two rec...

  17. Current Status of TRR-1/M1

    International Nuclear Information System (INIS)

    Sittichai, Chaiyut

    2000-01-01

    In 1961, the first Thai Research Reactor, TRR-1, having power of 1 MW was established. It was located at Office of Atomic Energy for Peace (OAEP) in Bangkok. TRR-1 was completely commissioned in June 1962. Plate typed high-enriched uranium (HEU) and U 3 O 8 -Al were used as fuel. Light water used as moderator and coolant. During 1975-1977, TRR-1 was shut down for modification. The reactor core and control system were disassembled and replaced by TRIGA Mark III. It is a circular hexagonal core typed reactor designed by General Atomics Company (GA). Afterwards, TRR-1 was officially renamed to Thai Research Reactor 1/Modification 1 (TRR-1/M1). TRR-1/M1 is a multipurpose reactor with nominal power of 2 MW. This swimming pool typed reactor uses low-enriched uranium (LEU) as fuel and light water as coolant and moderator. To date, the reactor has been operated with core No.12 that released power 1135 MWD to serve the user. The reactor has been serving for various kinds of utilization, for example, to produce radioisotope, neutron beam experiments and reactor physics experiments. This report explains in detail regarding operational experience and current status of this reactor, for example, reactor operation and reactor utilization. (author)

  18. Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Markus Thamm

    Full Text Available BACKGROUND: Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera, serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. METHODS: Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca(2+ imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. RESULTS: The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca(2+ concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. CONCLUSIONS: This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.

  19. Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera).

    Science.gov (United States)

    Thamm, Markus; Rolke, Daniel; Jordan, Nadine; Balfanz, Sabine; Schiffer, Christian; Baumann, Arnd; Blenau, Wolfgang

    2013-01-01

    Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca(2+) imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca(2+) concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.

  20. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    Science.gov (United States)

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

  2. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  3. Biological functionalization of drug delivery carriers to bypass size restrictions of receptor-mediated endocytosis independently from receptor targeting.

    Science.gov (United States)

    Ansar, Maria; Serrano, Daniel; Papademetriou, Iason; Bhowmick, Tridib Kumar; Muro, Silvia

    2013-12-23

    Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolae-mediated pathways, allows uptake of nano- and microcarriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and microcarriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems.

  4. Functional characterization of bursicon receptor and genome-wide analysis for identification of genes affected by bursicon receptor RNAi

    Science.gov (United States)

    Bai, Hua; Palli, Subba R.

    2010-01-01

    Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action. PMID:20457145

  5. Mood states, sympathetic activity, and in vivo beta-adrenergic receptor function in a normal population.

    Science.gov (United States)

    Yu, Bum-Hee; Kang, Eun-Ho; Ziegler, Michael G; Mills, Paul J; Dimsdale, Joel E

    2008-01-01

    The purpose of this study was to examine the relationship between mood states and beta-adrenergic receptor function in a normal population. We also examined if sympathetic nervous system activity is related to mood states or beta-adrenergic receptor function. Sixty-two participants aged 25-50 years were enrolled in this study. Mood states were assessed using the Profile of Mood States (POMS). Beta-adrenergic receptor function was determined using the chronotropic 25 dose isoproterenol infusion test. Level of sympathetic nervous system activity was estimated from 24-hr urine norepinephrine excretion. Higher tension-anxiety, depression-dejection, and anger-hostility were related to decreased beta-adrenergic receptor sensitivity (i.e., higher chronotropic 25 dose values), but tension-anxiety was the only remaining independent predictor of beta-adrenergic receptor function after controlling for age, gender, ethnicity, and body mass index (BMI). Urinary norepinephrine excretion was unrelated to either mood states or beta-adrenergic receptor function. These findings replicate previous reports that anxiety is related to decreased (i.e., desensitized) beta-adrenergic receptor sensitivity, even after controlling for age, gender, ethnicity, and body mass index.

  6. The structure and function of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Rasmussen, Søren Gøgsig Faarup; Kobilka, Brian K

    2009-01-01

    G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein...

  7. Receptor mutagenesis strategies for examination of structure-function relationships

    NARCIS (Netherlands)

    Blomenröhr, Marion; Vischer, Henry F; Bogerd, Jan

    2004-01-01

    This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base

  8. Scavenger receptor AI/II truncation, lung function and COPD

    DEFF Research Database (Denmark)

    Thomsen, M; Nordestgaard, B G; Tybjaerg-Hansen, A

    2011-01-01

    The scavenger receptor A-I/II (SRA-I/II) on alveolar macrophages is involved in recognition and clearance of modified lipids and inhaled particulates. A rare variant of the SRA-I/II gene, Arg293X, truncates the distal collagen-like domain, which is essential for ligand recognition. We tested whet...

  9. "m=1" coatings for neutron guides

    DEFF Research Database (Denmark)

    Cooper-Jensen, C.P.; Vorobiev, A.; Klinkby, Esben Bryndt

    2014-01-01

    A substantial part of the price for a neutron guide is the shielding needed because of the gamma ray produced when neutrons are absorbed. This absorption occurs in the coating and the substrate of the neutron guides. Traditional m=1 coatings have been made of Ni and if reflectivity over...... the critical angle of Ni is needed one has used Ni58 or Ni/Ti multilayer coatings. Ni has one of the highest neutron scattering density but it also has a fairly high absorption cross section for cold and thermal neutrons and when a neutron is absorbed it emits a lot of gamma rays, some with energies above 9 Me...... of diamond coatings to show the potential for using these coatings in neutron guides....

  10. E-ELT M1 test facility

    Science.gov (United States)

    Dimmler, M.; Marrero, J.; Leveque, S.; Barriga, P.; Sedghi, B.; Mueller, M.

    2012-09-01

    During the advanced design phase of the European Extremely Large Telescope (E-ELT) several critical components have been prototyped. During the last year some of them have been tested in dedicated test stands. In particular, a representative section of the E-ELT primary mirror has been assembled with 2 active and 2 passive segments. This test stand is equipped with complete prototype segment subunits, i.e. including support mechanisms, glass segments, edge sensors, position actuators as well as additional metrology for monitoring. The purpose is to test various procedures such as calibration, alignment and handling and to study control strategies. In addition the achievable component and subsystem performances are evaluated, and interface issues are identified. In this paper an overview of the activities related to the E-ELT M1 Test Facility will be given. Experiences and test results are presented.

  11. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  12. Functional antagonistic properties of clozapine at the 5-HT3 receptor.

    Science.gov (United States)

    Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

    1996-08-23

    The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

  13. Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors

    NARCIS (Netherlands)

    van den Akker, Emile; van Dijk, Thamar; Parren-van Amelsvoort, Martine; Grossmann, Katja S.; Schaeper, Ute; Toney-Earley, Kenya; Waltz, Susan E.; Löwenberg, Bob; von Lindern, Marieke

    2004-01-01

    Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the

  14. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  15. Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

    Science.gov (United States)

    Qureshi, O S; Rowley, T F; Junker, F; Peters, S J; Crilly, S; Compson, J; Eddleston, A; Björkelund, H; Greenslade, K; Parkinson, M; Davies, N L; Griffin, R; Pither, T L; Cain, K; Christodoulou, L; Staelens, L; Ward, E; Tibbitts, J; Kiessling, A; Smith, B; Brennan, F R; Malmqvist, M; Fallah-Arani, F; Humphreys, D P

    2017-12-06

    Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

  16. A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin*

    OpenAIRE

    Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D.; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

    2009-01-01

    Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepid...

  17. Analytic theory of the nonlinear M = 1 tearing mode

    International Nuclear Information System (INIS)

    Hazeltine, R.D.; Meiss, J.D.; Morrison, P.J.

    1985-09-01

    Numerical studies show that the m = 1 tearing mode continues to grow exponentially well into the nonlinear regime, in contrast with the slow, ''Rutherford,'' growth of m > 1 modes. We present a single helicity calculation which generalizes that of Rutherford to the case when the constant-psi approximation is invalid. As in that theory, the parallel current becomes an approximate flux function when the island size, W, exceeds the linear tearing layer width. However for the m = 1 mode, W becomes proportional to deltaB, rather than (deltaB)/sup 1/2/ above this critical amplitude. This implies that the convective nonlinearity in Ohm's law, which couples the m = 0 component to the m = 1 component, dominates the resistive diffusion term. The balance between the inductive electric field and this convective nonlinearity results in exponential growth. Assuming the form of the perturbed fields to be like that of the linear mode, we find that the growth occurs at 71% of the linear rate

  18. Helminthosporic acid functions as an agonist for gibberellin receptor.

    Science.gov (United States)

    Miyazaki, Sho; Jiang, Kai; Kobayashi, Masatomo; Asami, Tadao; Nakajima, Masatoshi

    2017-11-01

    Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure-activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

  19. Attenuated purinergic receptor function in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Thaning, Pia; Bune, Laurids T.; Hellsten, Ylva

    2010-01-01

    Objective: Extra cellular nucleotides and nucleosides are involved in regulation of skeletal muscle blood flow. Diabetes induces cardiovascular dysregulation but the extent to which the vasodilatatory capacity of nucleotides and nucleosides are affected in type 2 diabetes is unknown. The present...... study investigated: 1) the vasodilatatory effect of ATP, UTP, and adenosine (ADO) and 2) the expression and distribution of P2Y(2) and P2X(1) receptors in skeletal muscles of diabetic subjects. Research Design and Methods: In 10 diabetic patients and 10 age-matched controls, leg blood flow (LBF......-DM (1.5). The distribution and mRNA-expression of receptors were similar in the two groups. Conclusions: The vasodilatatory effect of the purinergic system is severely reduced in type 2 diabetic patients. The potency of nucleotides varies with the following rank order: UTP>ATP>>>ADO. This is not due...

  20. Insulin receptors

    International Nuclear Information System (INIS)

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the proceedings on insulin receptors. Part A: Methods for the study of structure and function. Topics covered include: Method for purification and labeling of insulin receptors, the insulin receptor kinase, and insulin receptors on special tissues

  1. A dynamic view of molecular switch behavior at serotonin receptors: implications for functional selectivity.

    Directory of Open Access Journals (Sweden)

    Maria Martí-Solano

    Full Text Available Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.

  2. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  3. Introduction to the m = 1 helicity source

    International Nuclear Information System (INIS)

    Platts, D.A.; Jarboe, T.R.; Wright, B.L.

    1985-01-01

    The m = 1 Helicity Source, formerly called the Kinked Z-pinch, was developed as part of the Electrode Studies program at Los Alamos. The Electrode Studies program was initiated to study the control of electrode erosion in long discharge duration spheromak sources. Erosion control is necessary to reduce plasma impurities and to obtain adequate electrode lifetimes. The first task of the Electrode Studies program is to determine, from among a variety of configurations including the coaxial one, a helicity source geometry with good prospects for erosion control. The more efficient the helicity source the easier it will be to control erosion, but the source most also be easy to diagnose and modify if it is to be a useful test bed. The various erosion control techniques which have been proposed will require extensive experimentation to evaluate and optimize. Proposed techniques include, using refractory metals, profiling of the electrodes and magnetic fields, and various gas injection schemes including porous electrodes. It is considered necessary to do these experiments on an optimized helicity source so that the electrode geometries and plasma properties will be relevant. Therefore the present Electrode Studies program is aimed at developing an improved helicity source design

  4. Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

    Science.gov (United States)

    Corin, Karolina; Baaske, Philipp; Ravel, Deepali B; Song, Junyao; Brown, Emily; Wang, Xiaoqiang; Wienken, Christoph J; Jerabek-Willemsen, Moran; Duhr, Stefan; Luo, Yuan; Braun, Dieter; Zhang, Shuguang

    2011-01-01

    A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

  5. Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

  6. Theory of the m=1 kink mode in toroidal plasmas

    International Nuclear Information System (INIS)

    Blank, J.H. de.

    1990-01-01

    The work in this thesis addresses the stability problems arising in tokamak experiments. In part I of this thesis the internal m=1 kink instability in tokamak plasmas is considered within the confines of ideal magnetohydrodynamics (ideal MHD), in which model the pressure is considered to be isotropic, while state is assumed. Because irreversible processes are disregarded, there is an energy principle. By extremizing the energy associated with infinitesimal perturbations of the plasma, a normal mode is obtained. The m=n=1 mode is resonant at the q=1 surface, and therefore, equilibria with a broad region where q#approx = # 1 are expected to be particularly unstable. The m=1 instability is computed for these q profiles. In Part II of this thesis, the internal m=1 kink instability is considered in a stationary rotating tokamak plasma, in which the particle velocity distribution is allowed to be non-thermal. In a tokamak plasma that is intensely heated by neutron beams or radiofrequent waves, these features, which cannot be described with ideal MHD, may become important, especially in the cases with high m=1 growth rates found in Part I. The energy principle of a generalized fluid theory is applied in these cases, without specifying the equation of state of the plasma. Therefore the resulting energy functional for the m=1 mode is incomplete and not direct applicable, however, the result makes clear that a kinetic description of the plasma is required only to a first approximation, and can therefore be applied analytically. The guiding center approximation is applied, which neglects finite gyroradius effects and collisions. Application of the dispersion relation, that is obtained from kinetic theory, shows that compared to the ideal MHD case, growth rates are strongly reduced due to Landau damping while the stability boundaries are not changed. (author). 72 refs.; 11 figs

  7. Functional specificity of sex pheromone receptors in the cotton bollworm Helicoverpa armigera.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available Male moths can accurately perceive the sex pheromone emitted from conspecific females by their highly accurate and specific olfactory sensory system. Pheromone receptors are of special importance in moth pheromone reception because of their central role in chemosensory signal transduction processes that occur in olfactory receptor neurons in the male antennae. There are a number of pheromone receptor genes have been cloned, however, only a few have been functionally characterized. Here we cloned six full-length pheromone receptor genes from Helicoverpa armigera male antennae. Real-time PCR showing all genes exhibited male-biased expression in adult antennae. Functional analyses of the six pheromone receptor genes were then conducted in the heterologous expression system of Xenopus oocytes. HarmOR13 was found to be a specific receptor for the major sex pheromone component Z11-16:Ald. HarmOR6 was equally tuned to both of Z9-16: Ald and Z9-14: Ald. HarmOR16 was sensitively tuned to Z11-16: OH. HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds. Our experiments elucidated the functions of some pheromone receptor genes of H. armigera. These advances may provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.

  8. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    Science.gov (United States)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  9. Regulation versus modulation in GnRH receptor function

    International Nuclear Information System (INIS)

    Zolman, J.C.; Theodoropoulos, T.J.

    1985-01-01

    Serum luteinizing hormone (LH) concentration after exposure to gonadotropin-releasing hormone (GnRH) indicates that an instantaneous increase occurs in the rate of release of LH directly from the anterior pituitary, as measured dynamically during superfusion in vitro. On the other hand, estradiol-17 beta (E2) alone shows no such instantaneous effect on LH release rate (at least for the first four hours), in either physiologic or pharmacologic concentrations. At the same time, brief (ten to 30 minute) exposure of isolated anterior pituitary plasma membranes to physiologic concentrations of E2 significantly alters the binding of a fully biologically active 125 I-GnRH to its plasma membrane receptor protein. In order to characterize the effect of E2 on GnRH binding further, dispersed bovine anterior pituitary cells were preincubated for six hours in the presence or absence of physiologic concentrations of E2 (10(-10)M). Following preincubation in the presence of E2, the cell suspension was incubated for 30 minutes with physiologic concentrations (5 x 10(-11) - 5 x 10(-10)M) of a fully biologically active 125 I-GnRH. The treatment, at least, doubled the number of biologically important high affinity GnRH binding sites (Kd's . 7.5 x -10(-11) - 4.5 x 10(-10)M), and changed the binding capacity of some of the binding sites up to three fold, which altered the cooperativity of GnRH-receptor interaction. Thus, the interaction of E2 with GnRH at the level of GnRH receptor is mandatory for the short-term pituitary effect of E2 on LH release in vitro and in vivo

  10. Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.

    Science.gov (United States)

    Desai, Swapna S; Roy, Binita Sur; Mahale, Smita D

    2013-12-01

    FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.

  11. Intrapulmonary receptors in the Tegu lizard: II. Functional characteristics and localization;.

    Science.gov (United States)

    Scheid, P; Kuhlmann, W D; Fedde, M R

    1977-02-01

    Intrapulmonary receptors identified in the Tegu lizard by single-unit vagal recording (Fedde et al., 1977) were subjected to a number of stimuli and localized within the lung. Some carbon dioxide receptors could follow periodic changes in intrapulmonary CO2 concentrations as rapidly as 1.3 Hz; No oxygen sensitivity was observed with this receptor type, and halothane markedly depressed the discharge frequency. In response to intravenously injected acetazolamide they increased their discharge frequency and became almost totally insensitive to CO2, suggesting molecular per se is not the direct controller of receptor discharge; These receptors show many of the functional characteristics described for those in the avian lung. Afferent activity from both CO2 and mechanoreceptors could be elicited by electrically stimulating the lung surface. The CO2 receptors appeared to be organized in a receptive field covering more than 1 cm2 of lung surface, multiple receptors being innervated by a single afferent fiber. Activity in afferent fibers from mechanoreceptors could be evoked from only one distinct spot on the lung surface. Conduction velocities of afferent fibers from CO2 receptors ranged from 1 to 3 m-sec-1; from mechanoreceptors, from 1.9 to 5.2 m-sec-1.

  12. Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

    Science.gov (United States)

    Yoon, SeongHun; Gariepy, Cheryl E; Yanagisawa, Masashi; Zuccarello, Mario; Rapoport, Robert M

    2016-03-01

    The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

  13. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  14. Expression and function of androgen receptor coactivator p44/Mep50/WDR77 in ovarian cancer.

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    Martin Ligr

    Full Text Available Hormones, including estrogen and progesterone, and their receptors play an important role in the development and progression of ovarian carcinoma. Androgen, its receptor and coactivators have also been implicated in these processes. p44/Mep50/WDR77 was identified as a subunit of the methylosome complex and lately characterized as a steroid receptor coactivator that enhances androgen receptor as well as estrogen receptor-mediated transcriptional activity in a ligand-dependent manner. We previously described distinct expression and function of p44 in prostate, testis, and breast cancers. In this report, we examined the expression and function of p44 in ovarian cancer. In contrast to findings in prostate and testicular cancer and similar to breast cancer, p44 shows strong cytoplasmic localization in morphologically normal ovarian surface and fallopian tube epithelia, while nuclear p44 is observed in invasive ovarian carcinoma. We observed that p44 can serve as a coactivator of both androgen receptor (AR and estrogen receptor (ER in ovarian cells. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in ovarian cancer cells in the presence of estrogen or androgen. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during ovarian tumorigenesis.

  15. Stabilization of functional recombinant cannabinoid receptor CB(2 in detergent micelles and lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Krishna Vukoti

    Full Text Available Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2, and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2, here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2 were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2 in dodecyl maltoside (DDM/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at

  16. Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

    DEFF Research Database (Denmark)

    Mi, Li-Zhi; Grey, Michael J; Nishida, Noritaka

    2008-01-01

    Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase...... differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane...

  17. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    International Nuclear Information System (INIS)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline; Sun, Jianmin; Jögi, Annika; Neumann, Drorit; Rönnstrand, Lars; Påhlman, Sven

    2014-01-01

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα + ) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells

  18. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  19. Agomelatine or ramelteon as treatment adjuncts in glioblastoma and other M1- or M2-expressing cancers.

    Science.gov (United States)

    Kast, Richard E

    2015-01-01

    The impressive but sad list of over forty clinical studies using various cytotoxic chemotherapies published in the last few years has failed to increase median survival of glioblastoma beyond two years after diagnosis. In view of this apparent brick wall, adjunctive non-cytotoxic growth factor blocking drugs are being tried, as in the CUSP9* protocol. A related theme is searching for agonists at growth inhibiting receptors. One such dataset is that of melatonin agonism at M1 or M2 receptors found on glioblastoma cells, being a negative regulator of these cells' growth. Melatonin itself is an endogenous hormone, but when used as an exogenously administered drug it has many disadvantages. Agomelatine, marketed as an antidepressant, and ramelteon, marketed as a treatment for insomnia, are currently-available melatonin receptor agonists. These melatonin receptor agonists have significant advantages over the natural ligand: longer half-life, better oral absorption, and higher affinity to melatonin receptors. They have an eminently benign side effect profile. As full agonists they should function to inhibit glioblastoma growth, as demonstrated for melatonin. A potentially helpful ancillary attribute of melatonergic agonists in glioblastoma treatment is an increase in interleukin-2 synthesis, expected, at least partially, to reverse some of the immunosuppression associated with glioblastoma.

  20. Functional pharmacology of cloned heterodimeric GABA-B receptors expressed in mammalian cells

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

    1999-01-01

    reported in different tissues, and this study thus provides a functional assay of cloned GABAB receptors which should be a valuable tool for further characterization of GABAB ligands. Finally, we can conclude that the functional pharmacological profiles of the two GABABR1 splice variants are very similar....

  1. METHODS FOR RECOMBINANT EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF HUMAN CANNABINOID RECEPTOR CB2

    Directory of Open Access Journals (Sweden)

    Alexei A. Yeliseev

    2013-03-01

    Full Text Available Cannabinoid receptor CB2 is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR. CB2 is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of CB2 suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant CB2 in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant CB2 receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.

  2. Cloning and functional characterization of the DA2 receptor gene in Chinese mitten crab (Eriocheir sinensis)

    Science.gov (United States)

    Xu, Min-jie; Zhang, Cong; Yang, Zhigang

    2018-01-01

    Dopamine (DA) plays a modulatory role in numerous physiological processes such as light adaptation and food intake, and exerts these functions through DA receptors (DARs). This study presents, for the first time, isolation and characterization of the dopamine receptor 2(DA2 receptor) cDNA from the intestinal tissue of Eriocheir sinensis, an economically important freshwater aquaculture species in China. The DA2 receptor cDNA sequence, which was obtained by rapid amplification of cDNA ends, is 2369bp long, encode peptide of 589 amino acid, and is highly homologous to related sequences in crustaceans. Analysis of the deduced amino acid sequence and the structure of the DA2 indicated that this receptor is a member of the family of G protein-coupled receptors (GPCRs), as it contains seven transmembrane domains and other common signatures of GPCRs. RT-PCR showed that the expression of the DA2 receptor gene was distributed in various tissues, and high expression levels were observed in the cranial ganglia and the thoracic ganglia. Further study of the effect of photoperiod on DA2 expression showed that constant darkness induced a significant increase in DA2 expression in the cranial ganglia. Finally, analysis of DA2 receptor expression under different feeding statuses showed that there was significantly greater expression in the hepatopancreas and intestines after feeding than before feeding, but there were no differences in expression between the before feeding and during feeding periods in either tissue. Our results indicate that the DA2 receptor structurally belongs to the family of G protein-coupled receptors, and that the cranial ganglia are the main tissues in which the DA2 receptor participates in light adaptation during dark hours. In addition, the DA2 receptor in E. sinensis may be involved in the physiological regulation of the hepatopancreas and digestive tract after the ingestion of food. This study provides a foundation for further exploration of the light

  3. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.

    Directory of Open Access Journals (Sweden)

    Thomas B Duguet

    2016-07-01

    Full Text Available Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the

  4. Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms.

    Directory of Open Access Journals (Sweden)

    Becky J Proskocil

    Full Text Available BACKGROUND: Epidemiological studies link organophosphorus pesticide (OP exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. CONCLUSIONS/SIGNIFICANCE: These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

  5. Ebselen has lithium-like effects on central 5-HT2A receptor function.

    Science.gov (United States)

    Antoniadou, I; Kouskou, M; Arsiwala, T; Singh, N; Vasudevan, S R; Fowler, T; Cadirci, E; Churchill, G C; Sharp, T

    2018-02-27

    Lithium's antidepressant action may be mediated by inhibition of inositol monophosphatase (IMPase), a key enzyme in G q protein coupled receptor signalling. Recently, the antioxidant agent ebselen was identified as an IMPase inhibitor. Here we investigated both ebselen and lithium in models of the 5-HT 2A receptor, a G q protein coupled receptor implicated in lithium's actions. 5-HT 2A receptor function was modelled in mice by measuring the behavioural (head-twitches) and cortical immediate early gene (IEG; Arc, c-fos and Erg2 mRNA) responses to 5-HT 2A receptor agonist administration. Ebselen and lithium were administered either acutely or chronically prior to assessment of 5-HT 2A receptor function. Given the SSRI augmenting action of lithium and 5-HT 2A antagonists, ebselen was also tested for this action by co-administration with the SSRI citalopram in microdialysis (extracellular 5-HT) experiments. Acute and repeated administration of ebselen inhibited behavioural and IEG responses to the 5-HT 2A receptor agonist DOI. Repeated lithium also inhibited DOI-evoked behavioural and IEG responses. In comparison, a selective IMPase inhibitor (L-690,330) attenuated the behavioural response to DOI whereas glycogen synthase kinase inhibitor (AR-A014418) did not. Finally, ebselen increased regional brain 5-HT synthesis and enhanced the increase in extracellular 5-HT induced by citalopram. The current data demonstrate lithium-mimetic effects of ebselen in different experimental models of 5-HT 2A receptor function, likely mediated by IMPase inhibition. This evidence of lithium-like neuropharmacological effects of ebselen adds further support for the clinical testing of ebselen in mood disorder, including as an antidepressant augmenting agent. This article is protected by copyright. All rights reserved.

  6. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

    2012-01-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  7. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Directory of Open Access Journals (Sweden)

    Venkateswarlu Kanamarlapudi

    Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  8. Differential compartmentalization and distinct functions of GABAB receptor variants

    DEFF Research Database (Denmark)

    Vigot, Réjan; Barbieri, Samuel; Bräuner-Osborne, Hans

    2006-01-01

    , while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved...... protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions....

  9. How membrane lipids control the 3D structure and function of receptors

    Directory of Open Access Journals (Sweden)

    Jacques Fantini

    2018-02-01

    Full Text Available The cohabitation of lipids and proteins in the plasma membrane of mammalian cells is controlled by specific biochemical and biophysical rules. Lipids may be either constitutively tightly bound to cell-surface receptors (non-annular lipids or less tightly attached to the external surface of the protein (annular lipids. The latter are exchangeable with surrounding bulk membrane lipids on a faster time scale than that of non-annular lipids. Not only do non-annular lipids bind to membrane proteins through stereoselective mechanisms, they can also help membrane receptors acquire (or maintain a functional 3D structure. Cholesterol is the prototype of membrane lipids that finely controls the 3D structure and function of receptors. However, several other lipids such as sphingolipids may also modulate the function of membrane proteins though conformational adjustments. All these concepts are discussed in this review in the light of representative examples taken from the literature.

  10. Xenobiotic Receptor-Mediated Regulation of Intestinal Barrier Function and Innate Immunity

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    Harmit S. Ranhotra

    2016-07-01

    Full Text Available The molecular basis for the regulation of the intestinal barrier is a very fertile research area. A growing body of knowledge supports the targeting of various components of intestinal barrier function as means to treat a variety of diseases, including the inflammatory bowel diseases. Herein, we will summarize the current state of knowledge of key xenobiotic receptor regulators of barrier function, highlighting recent advances, such that the field and its future are succinctly reviewed. We posit that these receptors confer an additional dimension of host-microbe interaction in the gut, by sensing and responding to metabolites released from the symbiotic microbiota, in innate immunity and also in host drug metabolism. The scientific evidence for involvement of the receptors and its molecular basis for the control of barrier function and innate immunity regulation would serve as a rationale towards development of non-toxic probes and ligands as drugs.

  11. Expression and purification of functional human mu opioid receptor from E.coli.

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    Yanbin Ma

    Full Text Available N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

  12. Structural basis and functions of abscisic acid receptors PYLs

    Science.gov (United States)

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  13. TLR receptors in laryngeal carcinoma - immunophenotypic, molecular and functional studies.

    Directory of Open Access Journals (Sweden)

    Mirosław Szczepański

    2011-04-01

    Full Text Available Toll-like receptors (TLRs have been shown to play crucial role in the recognition of unicellular pathogens. We have shown the expression of three TLRs on tumor cells of human laryngeal carcinoma by means of immunohistochemistry. In the current study we searched presence of TLR1-10 on protein and molecular level in larynx carcinoma cell lines and the impact of respective TLR ligands on TLR expression. Larynx carcinoma cell lines have been used. Cell were subjected to immunocytochemistry. RNA isolated from the cells was tested by RT-PCR. Cells were cultured in the presence of respective TLR ligands. Cells than were harvested and subjected to flow cytometry, using anti TLR1-10 Moabs. The cells were evaluated of membrane and cytoplasmic cell staining. TLR reactivity varied in individual cell lines. RT-PCR allowed to show mRNA for all TLRs tested. After short-term cell culture each cell line exhibited distinct pattern of expression of TLRs following interaction with respective ligand. Cytoplasmic TLR staining had usually higher MFI value than membrane one, but after culture with ligand it became reversed. TLRs 7 and 9 showed highest expression in the majority of tumor cells tested. In conclusion, larynx carcinoma cell lines exhibit rather universal expression of TLRs, both on protein and molecular level. Culture of TLR expressing tumor cells with ligands points out for potential reactivity of tumor cells with TLR agonists, what may have therapeutic implications.

  14. In vitro function of the aryl hydrocarbon receptor predicts in ...

    Science.gov (United States)

    Differences in sensitivity to dioxin-like compounds (DLCs) among species and taxa presents a major challenge to ecological risk assessments. Activation of the aryl hydrocarbon receptor (AHR) regulates adverse effects associated with exposure to DLCs in vertebrates. Prior investigations demonstrated that sensitivity to activation of the AHR1 (50% effect concentration; EC50) in an in vitro luciferase reporter gene (LRG) assay was predictive of the sensitivity of embryos (lethal dose to cause 50% lethality; LD50) across all species of birds for all DLCs. However, nothing was known about whether sensitivity to activation of the AHR is predictive of sensitivity of embryos of fishes to DLCs. Therefore, this study investigated in vitro sensitivities of AHR1s and AHR2s to the model DLC, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), among eight species of fish of known sensitivities of embryos to TCDD. AHR1s and AHR2s of all fishes were activated by TCDD in vitro. There was no significant linear relationship between in vitro sensitivity of AHR1 and in vivo sensitivity among the investigated fishes (R2 = 0.33, p = 0.23). However, there was a significant linear relationship between in vitro sensitivity of AHR2 and in vivo sensitivity among the investigated fishes (R2 = 0.97, p = fishes was compared to the previously generated linear relationship between in vitro s

  15. Characterization of Angiotensin II Molecular Determinants Involved in AT1 Receptor Functional Selectivity.

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J; Richard, Alexandra; Vandenberghe, Camille; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2015-06-01

    The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins Gq and G12, as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, βarrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and position 8 confers a bias toward βarrestin recruitment over ERK activation and Gq signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCζ and PKCι. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

    Science.gov (United States)

    Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

    2006-10-01

    To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

  17. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

    Directory of Open Access Journals (Sweden)

    Elise Courtot

    2015-12-01

    Full Text Available Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

  18. Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

    Directory of Open Access Journals (Sweden)

    Elodie Urlacher

    Full Text Available Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA and its receptor (Apime-ASTA-R; and C-type allatostatins (Apime-ASTC and Apime-ASTCC and their common receptor (Apime-ASTC-R. Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

  19. Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells.

    Science.gov (United States)

    Freund-Michel, Véronique; Frossard, Nelly

    2008-10-01

    Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

  20. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

    Science.gov (United States)

    Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

    2015-01-01

    Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

  1. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  2. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    International Nuclear Information System (INIS)

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the α-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced 22 Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the γ, δ, and ε subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four (α, β, γ, and δ) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the α, β, and δ subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the γ- and ε- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the γ is replaced by ε

  3. Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor.

    Science.gov (United States)

    Blenau, W; Balfanz, S; Baumann, A

    2000-03-01

    Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.

  4. Molecular Mechanisms Underlying the Link between Nuclear Receptor Function and Cholesterol Gallstone Formation

    Directory of Open Access Journals (Sweden)

    Mary Carmen Vázquez

    2012-01-01

    Full Text Available Cholesterol gallstone disease is highly prevalent in western countries, particularly in women and some specific ethnic groups. The formation of water-insoluble cholesterol crystals is due to a misbalance between the three major lipids present in the bile: cholesterol, bile salts, and phospholipids. Many proteins implicated in biliary lipid secretion in the liver are regulated by several transcription factors, including nuclear receptors LXR and FXR. Human and murine genetic, physiological, pathophysiological, and pharmacological evidence is consistent with the relevance of these nuclear receptors in gallstone formation. In addition, there is emerging data that also suggests a role for estrogen receptor ESR1 in abnormal cholesterol metabolism leading to gallstone disease. A better comprehension of the role of nuclear receptor function in gallstone formation may help to design new and more effective therapeutic strategies for this highly prevalent disease condition.

  5. 5α-Bile alcohols function as farnesoid X receptor antagonists

    International Nuclear Information System (INIS)

    Nishimaki-Mogami, Tomoko; Kawahara, Yosuke; Tamehiro, Norimasa; Yoshida, Takemi; Inoue, Kazuhide; Ohno, Yasuo; Nagao, Taku; Une, Mizuho

    2006-01-01

    The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5β-configuration in FXR activation. The results showed that the 5β-(A/B cis) bile alcohols 5β-cyprinol and bufol are potent FXR agonists, whereas their 5α-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A orientation of bile salts in agonist/antagonist function

  6. Application of 5-hydroxytryptamine receptor imaging for study of neuropsychiatric disorders and brain functions

    International Nuclear Information System (INIS)

    Qiu Chun; Guan Yihui

    2011-01-01

    In the central nervous system, the widely distributed 5-hydroxytryptamine (5-HT)receptors are involved in regulating a large number of psychological and physiological functions, including mood, sleep, endocrine and autonomic nervous system. Abnormal 5-HT transmission has been implicated in a variety of neuropsychiatric disorders, such as pain, depression and epilepsy. With the development of radioligands, non-invasive nuclear imaging technique with exquisite sensitivity and specificity has been applied for delineation of neurotransmitter function in vivo. It does great benefit for researches of these diseases and development of drugs. This review provided an overview of 5-HT receptors radioligands and recent findings. (authors)

  7. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

    Directory of Open Access Journals (Sweden)

    Darya V. Bazovkina

    2015-01-01

    Full Text Available In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors.

  8. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  9. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years.

    Directory of Open Access Journals (Sweden)

    Richard D Antrobus

    Full Text Available Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults.Thirty volunteers (aged 50-85 received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8 plaque forming units (pfu. Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP and matrix protein 1 (M1 was determined by interferon-gamma (IFN-γ ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+ T cells, T cell receptor (TCR gene expression was evaluated using an unbiased molecular approach.Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+ and CD8(+ T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+ T cells, which displayed a predominant CD27(+CD45RO(+CD57(-CCR7(- phenotype both before and after vaccination.MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination.ClinicalTrials.gov NCT00942071.

  10. A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin*

    Science.gov (United States)

    Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D.; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

    2009-01-01

    Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera. PMID:19416969

  11. Functional Studies of Sex Pheromone Receptors in Asian Corn Borer Ostrinia furnacalis

    Directory of Open Access Journals (Sweden)

    Wei Liu

    2018-05-01

    Full Text Available Lepidopteran insects use sex pheromones for sexual communication. Pheromone receptors expressed on peripheral olfactory receptor neurons (ORNs are critical part to detect the sex pheromones. In genus Ostrinia, several pheromone receptors were functional analyzed in O. nubilalis and O. scapulalis but the knowledge in O. furnacalis was rare. In this study, seven pheromone receptors were deorphanized by heterologous expression system of Xenopus oocytes. Functional types of sensilla trichoidea were classified by single sensillum recordings to interpret the response pattern of olfactory sensory neurons to Ostrinia pheromone components. OfurOR4 and OfurOR6 responded to the major sex pheromone Z/E12-14:OAc. OfurOR4 is the main receptor for both Z/E12-14:OAc and OfurOR6 mainly responded to E12-14:OAc. Functional differentiation of gene duplication were found between OfurOR5a and OfurOR5b. OfurOR5b showed a broad response to most of the pheromone components in O. furnacalis, whereas OfurOR5a was found without ligands. OfurOR7 showed a specific response to Z9-14:OAc and OfurOR8 mainly responded to Z11-14:OAc and E11-14:OAc. OfurOR3 did not respond to any pheromone components. Our results improved the current knowledge of pheromone reception in Ostrinia species which may contribute to speciation.

  12. CD147 Immunoglobulin Superfamily Receptor Function and Role in Pathology

    OpenAIRE

    Iacono, Kathryn T.; Brown, Amy L.; Greene, Mark I.; Saouaf, Sandra J.

    2007-01-01

    The immunoglobulin superfamily member CD147 plays an important role in fetal, neuronal, lymphocyte and extracellular matrix development. Here we review the current understanding of CD147 expression and protein interactions with regard to CD147 function and its role in pathologic conditions including heart disease, Alzheimer’s disease, stroke and cancer. A model linking hypoxic conditions found within the tumor microenvironment to up-regulation of CD147 expression and tumor progression is intr...

  13. Neto2 influences on kainate receptor pharmacology and function

    DEFF Research Database (Denmark)

    Han, Liwei; Howe, James; Pickering, Darryl S

    2016-01-01

    the mechanism of Neto2 modulation is still unclear, gain-of-function results from the characterization of GluK1-GluA2 chimeras indicate that the GluK1 sequences included in these chimeras (part or all of the TMD and part of the linkers between the TMDs and LBD) play a key role in Neto2 modulation of KAR...

  14. On the existence and function of galanin receptor heteromers in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Kjell eFuxe

    2012-10-01

    Full Text Available Galanin receptor (GalR subtypes1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein coupled receptors (GPCRs in the Central Nervous System (CNS. These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15 to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with transinhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15 in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15 but not galanin (1-29 can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression.

  15. Functional transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 channels along different segments of the renal vasculature

    DEFF Research Database (Denmark)

    Chen, L; Kaßmann, M; Sendeski, M

    2015-01-01

    with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.......AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized...... that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels. METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels. RESULTS: The TRPV1 agonist capsaicin relaxed mouse...

  16. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

    2018-03-28

    G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

  17. Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion.

    Science.gov (United States)

    Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E; Morales, Miguel A; Cifuentes, Fredy

    2017-01-01

    Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

  18. Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

    Directory of Open Access Journals (Sweden)

    Pablo Valle-Leija

    2017-07-01

    Full Text Available Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc, but are not thought to express a functional full-length TrkB receptor (TrkB-Fl. We, and others, have demonstrated that nerve growth factor (NGF and brain derived neurotrophic factor (BDNF modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR, 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

  19. Blockade of human P2X7 receptor function with a monoclonal antibody.

    Science.gov (United States)

    Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

    1998-11-15

    A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

  20. The Role of TAM Family Receptors in Immune Cell Function: Implications for Cancer Therapy.

    Science.gov (United States)

    Paolino, Magdalena; Penninger, Josef M

    2016-10-21

    The TAM receptor protein tyrosine kinases-Tyro3, Axl, and Mer-are essential regulators of immune homeostasis. Guided by their cognate ligands Growth arrest-specific gene 6 (Gas6) and Protein S (Pros1), these receptors ensure the resolution of inflammation by dampening the activation of innate cells as well as by restoring tissue function through promotion of tissue repair and clearance of apoptotic cells. Their central role as negative immune regulators is highlighted by the fact that deregulation of TAM signaling has been linked to the pathogenesis of autoimmune, inflammatory, and infectious diseases. Importantly, TAM receptors have also been associated with cancer development and progression. In a cancer setting, TAM receptors have a dual regulatory role, controlling the initiation and progression of tumor development and, at the same time, the associated anti-tumor responses of diverse immune cells. Thus, modulation of TAM receptors has emerged as a potential novel strategy for cancer treatment. In this review, we discuss our current understanding of how TAM receptors control immunity, with a particular focus on the regulation of anti-tumor responses and its implications for cancer immunotherapy.

  1. Sheep oocyte expresses leptin and functional leptin receptor mRNA

    Directory of Open Access Journals (Sweden)

    Seyyed Jalil Taheri

    2016-09-01

    Conclusions: The result of present study reveals that leptin and its functional receptor (Ob-Rb mRNA are expressed in sheep oocyte and further studies should investigate the role(s of leptin on sheep oocyte physiology and embryo development.

  2. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

    NARCIS (Netherlands)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

  3. Establishment of Sf9 transformants constitutively expressing PBAN receptor variants: application to functional evaluation

    Science.gov (United States)

    To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines stably expressing a number of fluorescent Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants incl...

  4. Common functional mineralocorticoid receptor polymorphisms modulate the cortisol awakening response : Interaction with SSRIs

    NARCIS (Netherlands)

    Klok, Melanie D.; Vreeburg, Sophie A.; Penninx, Brenda W. J. H.; Zitman, Frans G.; de Kloet, E. Ron; DeRijk, Roel H.

    Background: Cortisol controls the activity of the hypothalamic pituitary adrenal (HPA) axis during stress and during the circadian cycle through central mineralocorticoid (MR) and glucocorticoid receptors (GR). Changes in MR and GR functioning, therefore, may affect HPA axis activity. In this study

  5. Glutamate mediates the function of melanocortin receptor 4 on sim1 neurons in body weight regulation

    Science.gov (United States)

    The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing ...

  6. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

    Directory of Open Access Journals (Sweden)

    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  7. p75 Neurotrophin Receptor in the Skin: Beyond Its Neurotrophic Function.

    Science.gov (United States)

    Pincelli, Carlo

    2017-01-01

    p75 neurotrophin receptor (p75 NTR ), also known as CD271, is the low-affinity receptor that, together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), mediate neurotrophin (NT) functions. Beside their classic role in skin innervation, NT and their receptors constitute a complex cutaneous network associated with a number of autocrine and paracrine activities. In this context, the role of p75 NTR is becoming more and more important. This review will focus on the intriguing functions of p75 NTR in healthy and diseased skin. First, p75 NTR counterbalances the proliferative and survival activities of its cognate receptor Trk by inducing keratinocyte apoptosis. In addition, p75 NTR identifies an early transit-amplifying (TA) keratinocyte population and plays a critical role in keratinocyte stem cell transition to its progeny as well as in epidermal differentiation. p75 NTR is absent in psoriatic TA cells, thus rendering these cells resistant to apoptosis. On the other hand, p75 NTR infection restores NT-induced apoptosis in psoriatic keratinocytes. Taken together, these results provide evidence for a critical role of p75 NTR in epidermal homeostasis, while its lack may account for the TA defect in psoriasis. While the issue of p75 NTR as a marker of melanoma initiating cells is still to be solved, there is strong evidence that downregulation of this receptor is a precondition to melanoma invasion and metastasis in vitro and in vivo . All in all, this review points to p75 NTR as a major actor in both physiologic and pathologic conditions at the skin level.

  8. Aberrant dopamine D2-like receptor function in a rodent model of schizophrenia.

    Science.gov (United States)

    Perez, Stephanie M; Lodge, Daniel J

    2012-11-01

    Based on the observation that antipsychotic medications display antagonist properties at dopamine D2-like receptors, aberrant dopamine signaling has been proposed to underlie psychosis in patients with schizophrenia. Thus, it is not surprising that considerable research has been devoted to understanding the mechanisms involved in the antipsychotic action of these compounds. It is important to note that the majority of these studies have been performed in "normal" experimental animals. Given that these animals do not possess the aberrant neuronal information processing typically associated with schizophrenia, the aim of the current study was to examine the dopamine D2 receptor system in a rodent model of schizophrenia. Here, we demonstrate that methylazoxymethanol acetate (MAM)-treated rats display an enhanced effect of quinpirole on dopamine neuron activity and an aberrant locomotor response to D2-like receptor activation, suggesting changes in postsynaptic D2-like receptor function. To better understand the mechanisms underlying the enhanced response to D2-like ligands in MAM-treated rats, we examined the expression of D2, D3, and dopamine transporter mRNA in the nucleus accumbens and ventral tegmental area by quantitative reverse transcription-polymerase chain reaction. MAM-treated rats displayed a significant increase in dopamine D3 receptor mRNA expression in the nucleus accumbens with no significant changes in the expression of the D2 receptor. Taken together, these data demonstrate robust alterations in dopamine D2-like receptor function in a rodent model of schizophrenia and provide evidence that preclinical studies examining the mechanisms of antipsychotic drug action should be performed in animal models that mirror aspects of the abnormal neuronal transmission thought to underlie symptoms of schizophrenia.

  9. Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

    Directory of Open Access Journals (Sweden)

    Harbison Carole E

    2007-02-01

    Full Text Available Abstract Background Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS, and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV. Feline aminopeptidase N (fAPN serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV, canine coronavirus, transmissible gastroenteritis virus (TGEV, and human coronavirus 229E (HCoV-229E. A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056. Results Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41, but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 ( Conclusion We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

  10. Structural and functional plasticity of the luteinizing hormone/choriogonadotrophin receptor.

    Science.gov (United States)

    Troppmann, Britta; Kleinau, Gunnar; Krause, Gerd; Gromoll, Jörg

    2013-01-01

    BACKGROUND In recent years it became evident that several types of the luteinizing hormone/choriogonadotrophin receptor (LHCGR) exist. In addition to the classical receptor type known in rodents, an LHCGR type containing an additional exon is present in primates and humans. This specific exon 6A introduces a hitherto unknown regulatory pathway of the LHCGR at the transcriptional level which can lead to the expression of an alternative protein covering the extracellular part only. Furthermore, an LHCGR type lacking exon 10 at the mRNA and protein levels has been described in the New World primate lineage, giving rise to an additional receptor type in which amino acids of the extracellular hinge region connecting the leucine-rich repeat domain and transmembrane domain are missing. METHODS Topic-related information was retrieved by systematic searches using Medline/PubMed. Structural homology models were retrieved from a glycoprotein hormone receptors web application and from recent publications. RESULTS In a novel approach, we combine functional aspects with three-dimensional properties of the LHCGR and the different receptor types to deduce causative relationships between these two parameters. On this basis, the physiological impact and patho-physiological consequences of the different LHCGR types are inferred. CONCLUSIONS The complex system of different LHCGR types and two corresponding hormones (LH and CG) represents a major challenge for future studies on selective hormone binding, signal transduction and receptor regulation. The presence of these naturally occurring LHCGR types requires re-examining of our present view on receptor function, experimental set-ups and data interpretation, but also offers new clinical approaches to interfere with LH/CG action in humans.

  11. The structure and function of glutamate receptors: Mg2+ block to X-ray diffraction.

    Science.gov (United States)

    Mayer, Mark L

    2017-01-01

    Experiments on the action of glutamate on mammalian and amphibian nervous systems started back in the 1950s but decades passed before it became widely accepted that glutamate was the major excitatory neurotransmitter in the CNS. The pace of research greatly accelerated in the 1980s when selective ligands that identified glutamate receptor subtypes became widely available, and voltage clamp techniques, coupled with rapid perfusion, began to resolve the unique functional properties of what cloning subsequently revealed to be a large family of receptors with numerous subtypes. More recently the power of X-ray crystallography and cryo-EM has been applied to the study of glutamate receptors, revealing their atomic structures, and the conformational changes that underlie their gating. In this review I summarize the history of this field, viewed through the lens of a career in which I spent 3 decades working on the structure and function of glutamate receptor ion channels. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'. Published by Elsevier Ltd.

  12. Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients.

    Directory of Open Access Journals (Sweden)

    Andreas Binder

    Full Text Available Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K was associated with the presence of paradoxical heat sensation (p = 0.03, and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V with cold hypoalgesia (p = 0.0035. Two main subgroups characterized by preserved (1 and impaired (2 sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and pG (rs222747, M315I to cold hypaesthesia (p = 0.002, but there was absence of associations in subgroup 2. In this study we found no evidence that genetic

  13. Infinitely many N=1 dualities from m+1m=1

    Energy Technology Data Exchange (ETDEWEB)

    Agarwal, Prarit; Intriligator, Kenneth; Song, Jaewon [Department of Physics, University of California,San Diego, La Jolla, CA 92093 (United States)

    2015-10-06

    We discuss two infinite classes of 4d supersymmetric theories, T{sub N}{sup (m)} and U{sub N}{sup (m)}, labelled by an arbitrary non-negative integer, m. The T{sub N}{sup (m)} theory arises from the 6d, A{sub N−1} type N=(2,0) theory reduced on a 3-punctured sphere, with normal bundle given by line bundles of degree (m+1,−m); the m=0 case is the N=2 supersymmetric T{sub N} theory. The novelty is the negative-degree line bundle. The U{sub N}{sup (m)} theories likewise arise from the 6d N=(2,0) theory on a 4-punctured sphere, and can be regarded as gluing together two (partially Higgsed) T{sub N}{sup (m)} theories. The T{sub N}{sup (m)} and U{sub N}{sup (m)} theories can be represented, in various duality frames, as quiver gauge theories, built from T{sub N} components via gauging and nilpotent Higgsing. We analyze the RG flow of the U{sub N}{sup (m)} theories, and find that, for all integer m>0, they end up at the same IR SCFT as SU(N) SQCD with 2N flavors and quartic superpotential. The U{sub N}{sup (m)} theories can thus be regarded as an infinite set of UV completions, dual to SQCD with N{sub f}=2N{sub c}. The U{sub N}{sup (m)} duals have different duality frame quiver representations, with 2m+1 gauge nodes.

  14. Functional Analysis of the Tomato Immune Receptor Ve1 through Domain Swaps with Its Non-Functional Homolog Ve2

    Science.gov (United States)

    Rovenich, Hanna; Song, Yin; Liebrand, Thomas W. H.; Masini, Laura; van den Berg, Grardy C. M.; Joosten, Matthieu H. A. J.; Thomma, Bart P. H. J.

    2014-01-01

    Resistance in tomato against race 1 strains of the fungal vascular wilt pathogens Verticillium dahliae and V. albo-atrum is mediated by the Ve locus. This locus comprises two closely linked inversely oriented genes, Ve1 and Ve2, which encode cell surface receptors of the extracellular leucine-rich repeat receptor-like protein (eLRR-RLP) type. While Ve1 mediates Verticillium resistance through monitoring the presence of the recently identified V. dahliae Ave1 effector, no functionality for Ve2 has been demonstrated in tomato. Ve1 and Ve2 contain 37 eLRRs and share 84% amino acid identity, facilitating investigation of Ve protein functionality through domain swapping. In this study it is shown that Ve chimeras in which the first thirty eLRRs of Ve1 were replaced by those of Ve2 remain able to induce HR and activate Verticillium resistance, and that deletion of these thirty eLRRs from Ve1 resulted in loss of functionality. Also the region between eLRR30 and eLRR35 is required for Ve1-mediated resistance, and cannot be replaced by the region between eLRR30 and eLRR35 of Ve2. We furthermore show that the cytoplasmic tail of Ve1 is required for functionality, as truncation of this tail results in loss of functionality. Moreover, the C-terminus of Ve2 fails to activate immune signaling as chimeras containing the C-terminus of Ve2 do not provide Verticillium resistance. Furthermore, Ve1 was found to interact through its C-terminus with the eLRR-containing receptor-like kinase (eLRR-RLK) interactor SOBIR1 that was recently identified as an interactor of eLRR-RLP (immune) receptors. Intriguingly, also Ve2 was found to interact with SOBIR1. PMID:24505431

  15. Anxiety and beta-adrenergic receptor function in a normal population.

    Science.gov (United States)

    Kang, Eun-Ho; Yu, Bum-Hee

    2005-06-01

    Many studies have shown a close relationship between anxiety and beta-adrenergic receptor function in patients with anxiety disorders. This study examined the relationship between beta-adrenergic receptor function and anxiety levels in a normal population. Subjects for this study included 36 men and 44 women between the ages of 20 and 40 years whose Body Mass Index (BMI) was between 18 and 26. All of them were healthy subjects who had no previous history of medical or psychiatric illnesses. The authors measured the Spielberger State-Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), and Chronotropic 25 Dose (CD25) of isoproterenol, previously developed to assess in vivo beta-adrenergic receptor sensitivity. We also examined correlations between log normalized CD25 and mood states. The mean of CD25 was 2.64+/-1.37 mug and the mean of CD25 in men was significantly higher (i.e., lower beta-adrenergic receptor sensitivity) than that of women (3.26+/-1.35 vs. 2.14+/-1.17 microg; t = 3.99, p anxiety (r = -0.344, p = 0.002), trait anxiety (r = -0.331, p = 0.003), and BDI (r = -0.283, p = 0.011). CD25 was positively correlated with BMI (r = 0.423, p anxiety, and BMI. The sensitivity of beta-adrenergic receptors increased as anxiety levels became higher in a normal population. Thus, the relationship between anxiety and beta-adrenergic receptor function in healthy subjects may be different from that of patients with anxiety disorders.

  16. Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

    Science.gov (United States)

    Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

    2014-01-01

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

  17. Characterization of the functional domains of the natriuretic peptide receptor/guanylate cyclase by radiation inactivation

    International Nuclear Information System (INIS)

    Tremblay, J.; Huot, C.; Koch, C.; Potier, M.

    1991-01-01

    Radiation inactivation has been used to evaluate the molecular size of domains responsible for atrial natriuretic peptide (ANP)-binding and cyclase functions of the ANP receptor/guanylate cyclase. Two types of inactivation curves were observed for cyclase function in both adrenal cortex and aortic smooth muscle cells: (1) biphasic with enhanced guanylate cyclase activity after exposure to low radiation doses and (2) linear after preincubation of membrane proteins with 0.5 microM ANP or solubilization with Triton X-100. The existence of an inhibitory component was the simplest model that best explained the types of radiation curves obtained. Activation of guanylate cyclase by ANP or Triton X-100 could occur via the dissociation of this inhibitory component from the catalytic domain. On the other hand, the loss of ANP-binding activity was linear with increasing radiation exposures under basal, ANP treatment, and Triton X-100 solubilization conditions. Radiation inactivation sizes of about 30 kDa for cyclase function, 20 kDa for ANP-binding function, and 90 kDa for inhibitory function were calculated. These studies suggest that the ANP receptor/guanylate cyclase behaves as a multidomain protein. The results obtained by radiation inactivation of the various biological functions of this receptor are compatible with the hypothesis of an intramolecular inhibitory domain repressing the guanylate cyclase catalytic domain within its membrane environment

  18. Glucocorticoid-Induced Fetal Programming Alters the Functional Complement of Angiotensin Receptors Subtypes within the Kidney

    OpenAIRE

    Gwathmey, TanYa M.; Shaltout, Hossam A.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

    2011-01-01

    We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80–81 days gestation with full term delivery. Renal nuclear and plasma membrane fractions were isolated from 1.0–1.5 year old sheep for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were sign...

  19. Pharmacologic perspectives of functional selectivity by the angiotensin II type 1 receptor

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Hansen, Jakob Lerche

    2008-01-01

    and to sudden injury occurring in the circulatory system. Hence, current drugs that block all AT(1) receptor actions most likely leave room for improvement. Recent developments show that two major signaling pathways used by the AT(1) receptor may be dissected by pharmacologic means. Key pathologic responses...... protein actions and simultaneous activation of G protein-dependent or -independent signaling could therefore be desirable in certain situations. The previously unappreciated concept of "functional selectivity" makes this exact strategy feasible and may yield improved drugs for cardiovascular therapy....

  20. TAM receptor tyrosine kinase function and the immunopathology of liver disease.

    Science.gov (United States)

    Mukherjee, S K; Wilhelm, A; Antoniades, C G

    2016-06-01

    Tyro3, Axl, MERTK (TAM) receptor tyrosine kinases are implicated in the regulation of the innate immune response through clearance of apoptotic cellular debris and control of cytokine signaling cascades. As a result they are pivotal in regulating the inflammatory response to tissue injury. Within the liver, immune regulatory signaling is employed to prevent the overactivation of innate immunity in response to continual antigenic challenge from the gastrointestinal tract. In this review we appraise current understanding of the role of TAM receptor function in the regulation of both innate and adaptive immunity, with a focus on its impact upon hepatic inflammatory pathology. Copyright © 2016 the American Physiological Society.

  1. Heat-Labile Enterotoxin: Beyond G M1 Binding

    Directory of Open Access Journals (Sweden)

    Benjamin Mudrak

    2010-06-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside GM1, the toxin’s host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

  2. M1 transitions in the (sdg) boson model

    International Nuclear Information System (INIS)

    Kuyucak, S.; Tuebingen Univ.; Morrison, I.

    1988-01-01

    Using the 1/N expansion technique we derive expressions for β → g, γ → g and γ → γ M1 transitions in a general boson model. The M1 matrix elements in the sdg-boson model are similar in form to those in the neutron-proton IBM. Comparisons are made to some selected M1 data exhibiting collective character. (orig.)

  3. M1 transitions in the (sdg) boson model

    Energy Technology Data Exchange (ETDEWEB)

    Kuyucak, S.; Morrison, I.

    1988-03-03

    Using the 1/N expansion technique we derive expressions for ..beta.. -> g, ..gamma.. -> g and ..gamma.. -> ..gamma.. M1 transitions in a general boson model. The M1 matrix elements in the sdg-boson model are similar in form to those in the neutron-proton IBM. Comparisons are made to some selected M1 data exhibiting collective character.

  4. Stochasticity and the m = 1 mode in tokamaks

    International Nuclear Information System (INIS)

    Izzo, R.; Monticello, D.A.; Stodiek, W.; Park, W.

    1986-05-01

    It has recently been proposed that stochasticity resulting from toroidal coupling could lead to a saturation of the m = 1 internal mode in tokamaks. We present results from the nonlinear evolution of the m = 1 mode with toroidal coupling that show that stochasticity is not enough to cause saturation of the m = 1 mode

  5. S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2009-01-01

    Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

  6. Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils.

    Science.gov (United States)

    Tamaki, Mami; Konno, Yasunori; Kobayashi, Yoshiki; Takeda, Masahide; Itoga, Masamichi; Moritoki, Yuki; Oyamada, Hajime; Kayaba, Hiroyuki; Chihara, Junichi; Ueki, Shigeharu

    2014-07-01

    Sexual dimorphism in asthma links the estrogen and allergic immune responses. The function of estrogen was classically believed to be mediated through its nuclear receptors, i.e., estrogen receptors (ERs). However, recent studies established the important roles of G-protein-coupled estrogen receptor (GPER/GPR30) as a novel membrane receptor for estrogen. To date, the role of GPER in allergic inflammation is poorly understood. The purpose of this study was to examine whether GPER might affect the functions of eosinophils, which play an important role in the pathogenesis of asthma. Here, we demonstrated that GPER was expressed in purified human peripheral blood eosinophils both at the mRNA and protein levels. Although GPER agonist G-1 did not induce eosinophil chemotaxis or chemokinesis, preincubation with G-1 enhanced eotaxin (CCL11)-directed eosinophil chemotaxis. G-1 inhibited eosinophil spontaneous apoptosis and caspase-3 activities. The anti-apoptotic effect was not affected by the cAMP-phospodiesterase inhibitor rolipram or phosphoinositide 3-kinase inhibitors. In contrast to resting eosinophils, G-1 induced apoptosis and increased caspase-3 activities when eosinophils were co-stimulated with IL-5. No effect of G-1 was observed on eosinophil degranulation in terms of release of eosinophil-derived neurotoxin (EDN). The current study indicates the functional capacities of GPER on human eosinophils and also provides the previously unrecognized mechanisms of interaction between estrogen and allergic inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies

    DEFF Research Database (Denmark)

    Staus, Dean P; Wingler, Laura M; Strachan, Ryan T

    2014-01-01

    . However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate...... their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed...... on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies...

  8. Functional interactions between 7TM receptors in the renin-angiotensin system--dimerization or crosstalk?

    DEFF Research Database (Denmark)

    Lyngsø, Christina; Erikstrup, Niels; Hansen, Jakob L

    2008-01-01

    . The importance of the RAS is clearly emphasised by the widespread use of drugs targeting this system in clinical practice. These include, renin inhibitors, angiotensin II receptor type I blockers, and inhibitors of the angiotensin converting enzyme. Some of the important effectors within the system are 7...... be important for receptor function, and in the development of cardiovascular diseases. This is very significant, since "dimers" may provide pharmacologists with novel targets for improved drug therapy. However, we know that 7TM receptors can mediate signals as monomeric units, and so far it has been very......The Renin-Angiotensin System (RAS) is important for the regulation of cardiovascular physiology, where it controls blood pressure, and salt- and water homeostasis. Dysregulation of RAS can lead to severe diseases including hypertension, diabetic nephropathy, and cardiac arrhythmia, and -failure...

  9. Neurovascular coupling to D2/D3 dopamine receptor occupancy using simultaneous PET/functional MRI

    DEFF Research Database (Denmark)

    Sander, Christin Y; Hooker, Jacob M; Catana, Ciprian

    2013-01-01

    This study employed simultaneous neuroimaging with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to demonstrate the relationship between changes in receptor occupancy measured by PET and changes in brain activity inferred by fMRI. By administering the D2/D3...... dopamine receptor antagonist [(11)C]raclopride at varying specific activities to anesthetized nonhuman primates, we mapped associations between changes in receptor occupancy and hemodynamics [cerebral blood volume (CBV)] in the domains of space, time, and dose. Mass doses of raclopride above tracer levels...... caused increases in CBV and reductions in binding potential that were localized to the dopamine-rich striatum. Moreover, similar temporal profiles were observed for specific binding estimates and changes in CBV. Injection of graded raclopride mass doses revealed a monotonic coupling between neurovascular...

  10. Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney.

    Science.gov (United States)

    Gwathmey, TanYa M; Shaltout, Hossam A; Rose, James C; Diz, Debra I; Chappell, Mark C

    2011-03-01

    We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(1) receptors sensitive to losartan was 2-fold higher (67 ± 6% vs 27 ± 9%; Psheep (16 ± 3% vs 6 ± 4%; Pfetal programming.

  11. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

  12. Effect of genetic and pharmacological blockade of GABA receptors on the 5-HT2C receptor function during stress.

    OpenAIRE

    Martin Cédric B P; Gassmann Martin; Chevarin Caroline; Hamon Michel; Rudolph Uwe; Bettler Bernhard; Lanfumey Laurence; Mongeau Raymond

    2014-01-01

    5-HT2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on serotonin (5-HT) release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5-HT2C receptor-induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5-HT tur...

  13. [Functional properties of taste bud cells. Mechanisms of afferent neurotransmission in Type II taste receptor cells].

    Science.gov (United States)

    Romanov, R A

    2013-01-01

    Taste Bud cells are heterogeneous in their morphology and functionality. These cells are responsible for sensing a wide variety of substances and for associating detected compounds with a different taste: bitter, sweet, salty, sour and umami. Today we know that each of the five basic tastes corresponds to distinct cell populations organized into three basic morpho-functional cell types. In addition, some receptor cells of the taste bud demonstrate glia-related functions. In this article we expand on some properties of these three morphological receptor cell types. Main focus is devoted to the Type II cells and unusual mechanism for afferent neurotransmission in these cells. Taste cells of the Type II consist of three populations detecting bitter, sweet and umami tastes, and, thus, evoke a serious scientific interest.

  14. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

    Directory of Open Access Journals (Sweden)

    Hafner Martin

    2004-08-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. Results Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. Conclusion Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance.

  15. Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

    Directory of Open Access Journals (Sweden)

    Stefan Gahbauer

    2016-10-01

    Full Text Available The dimerization or even oligomerization of G protein coupled receptors (GPCRs causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.

  16. Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra

    International Nuclear Information System (INIS)

    Dawson, T.M.; Dawson, V.L.; Gage, F.H.; Fisher, L.J.; Hunt, M.A.; Wamsley, J.K.

    1991-01-01

    Interruption of the ascending dopamine neurons of the nigrostriatal pathway, by 6-hydroxydopamine (6-OHDA) lesion in rats, produced a significant loss of the dopamine transport complexes labeled with the phencyclidine derivative [3H]BTCP. This loss of dopamine innervation in the striatum was present at least 12 to 14 months after lesioning and was functionally manifested by ipsilateral rotation of the animals in response to amphetamine. In these same animals, in comparison to controls, there was a significant increase in the number (Bmax) of [3H]SCH 23390-labeled D-1 receptors in the striatum (36.7%) and the substantia nigra (35.1%) and a 54.4% increase in the number (Bmax) of [3H]sulpiride-labeled striatal D-2 receptors without an apparent change in affinity (Kd). Ten to twelve months after the transplantation of homologous fetal substantia nigra into the denervated striatum, there was a significant decrease in amphetamine-induced turning behavior. In these animals, there was an ingrowth of dopamine nerve terminals in the striatum as demonstrated by a return of [3H]BTCP binding. Accompanying this reinnervation was the normalization of D-1 and D-2 receptors to control values in the striatum as well as the return of D-1 receptors to prelesion densities in the substantia nigra. In a subgroup of transplanted rats, amphetamine continued to induce ipsilateral turning. In these animals both D-1 and D-2 receptors remained supersensitive. These results support the hypothesis that the functional recovery of transplanted animals is due, in part, to reinnervation of the striatum. In addition, long-term alterations in receptor density may be related to the behavioral deficits that are associated with the 6-OHDA-lesioned rat

  17. Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

    Science.gov (United States)

    Fischer, J A; Muff, R; Born, W

    2002-08-01

    The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

  18. The VPAC1 receptor: structure and function of a class B GPCR prototype

    Directory of Open Access Journals (Sweden)

    Alain eCouvineau

    2012-11-01

    Full Text Available The class B G protein-coupled receptors (GPCRs represents a small sub-family encompassing 15 members, and are very promising targets for the development of drugs to treat many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. The VPAC1 receptor which is an archetype of the class B GPCRs binds Vasoactive Intestinal Peptide (VIP, a neuropeptide widely distributed in central and peripheral nervous system modulating many physiological processes including regulation of exocrine secretions, hormone release, foetal development, immune response... VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC1 receptors. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted of VPAC1 plays a pivotal role in VIP recognition. The use of different approaches such as directed mutagenesis, photoaffinity labeling, Nuclear Magnetic Resonance (NMR, molecular modeling and molecular dynamic simulation has led to demonstrate that: i the central and C-terminal part of the VIP molecule interacts with the N-ted of VPAC1 receptor which is itself structured as a « Sushi » domain; ii the N-terminal end of the VIP molecule interacts with the first transmembrane domain of the receptor where three residues (K143, T144 and T147 play an important role in VPAC1 interaction with the first histidine residue of VIP.

  19. Small leucine zipper protein functions as a negative regulator of estrogen receptor α in breast cancer.

    Directory of Open Access Journals (Sweden)

    Juyeon Jeong

    Full Text Available The nuclear transcription factor estrogen receptor α (ERα plays a critical role in breast cancer progression. ERα acts as an important growth stimulatory protein in breast cancer and the expression level of ERα is tightly related to the prognosis and treatment of patients. Small leucine zipper protein (sLZIP functions as a transcriptional cofactor by binding to various nuclear receptors, including glucocorticoid receptor, androgen receptor, and peroxisome proliferator-activated receptor γ. However, the role of sLZIP in the regulation of ERα and its involvement in breast cancer progression is unknown. We found that sLZIP binds to ERα and represses the transcriptional activity of ERα in ERα-positive breast cancer cells. sLZIP also suppressed the expression of ERα target genes. sLZIP disrupted the binding of ERα to the estrogen response element of the target gene promoter, resulting in suppression of cell proliferation. sLZIP is a novel co-repressor of ERα, and plays a negative role in ERα-mediated cell proliferation in breast cancer.

  20. Evidence of changes in alpha-1/AT1 receptor function generated by diet-induced obesity.

    Science.gov (United States)

    Juarez, Esther; Tufiño, Cecilia; Querejeta, Enrique; Bracho-Valdes, Ismael; Bobadilla-Lugo, Rosa A

    2017-11-01

    To study whether hypercaloric diet-induced obesity deteriorates vascular contractility of rat aorta through functional changes in α 1 adrenergic and/or AT1 Angiotensin II receptors. Angiotensin II- or phenylephrine-induced contraction was tested on isolated aorta rings with and without endothelium from female Wistar rats fed for 7 weeks with hypercaloric diet or standard diet. Vascular expression of Angiotensin II Receptor type 1 (AT1R), Angiotensin II Receptor type 2 (AT2R), Cyclooxygenase-1 (COX-1), Cyclooxygenase-2 (COX-2), inducible Nitric Oxide Synthase (iNOS) and endothelial Nitric Oxide Synthase (eNOS), as well as blood pressure, glucose, insulin and angiotensin II blood levels were measured. Diet-induced obesity did not significantly change agonist-induced contractions (Emax and pD 2 hypercaloric diet vs standard diet n.s.d.) of both intact (e+) or endothelium free (e-) vessels but significantly decrease both phenylephrine and angiotensin II contraction (Emax p obesity did not change angiotensin II AT1, AT2 receptor proteins expression but reduced COX-1 and NOS2 ( p obesity produces alterations in vascular adrenergic and angiotensin II receptor dynamics that suggest an endothelium-dependent adrenergic/angiotensin II crosstalk. These changes reflect early-stage vascular responses to obesity.

  1. Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

    Directory of Open Access Journals (Sweden)

    Makiko Suwa

    2011-04-01

    Full Text Available An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS http://sevens.cbrc.jp/ that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

  2. Functional characteristics of the naked mole rat μ-opioid receptor.

    Directory of Open Access Journals (Sweden)

    Melanie Busch-Dienstfertig

    Full Text Available While humans and most animals respond to µ-opioid receptor (MOR agonists with analgesia and decreased aggression, in the naked mole rat (NMR opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1 can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3'-5'-cyclic adenosine monophosphate (cAMP enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR's extreme reaction to opioids.

  3. Expression and function of serotonin 2A and 2B receptors in the mammalian respiratory network.

    Directory of Open Access Journals (Sweden)

    Marcus Niebert

    Full Text Available Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT(2A, 5-HT(2B, and 5-HT(2C receptors that are directed towards protein kinase C (PKC. In contrast to 5-HT(2ARs, expression and function of 5-HT(2BRs within the respiratory network are still unclear. 5-HT(2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT(2ARs and 5-HT(2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT(2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation.

  4. Orexin Receptor Multimerization versus Functional Interactions: Neuropharmacological Implications for Opioid and Cannabinoid Signalling and Pharmacogenetics

    Directory of Open Access Journals (Sweden)

    Miles D. Thompson

    2017-10-01

    Full Text Available Orexins/hypocretins are neuropeptides formed by proteolytic cleavage of a precursor peptide, which are produced by neurons found in the lateral hypothalamus. The G protein-coupled receptors (GPCRs for these ligands, the OX1 and OX2 orexin receptors, are more widely expressed throughout the central nervous system. The orexin/hypocretin system has been implicated in many pathways, and its dysregulation is under investigation in a number of diseases. Disorders in which orexinergic mechanisms are being investigated include narcolepsy, idiopathic sleep disorders, cluster headache and migraine. Human narcolepsy has been associated with orexin deficiency; however, it has only rarely been attributed to mutations in the gene encoding the precursor peptide. While gene variations within the canine OX2 gene hcrtr2 have been directly linked with narcolepsy, the majority of human orexin receptor variants are weakly associated with diseases (the idiopathic sleep disorders, cluster headache and polydipsia-hyponatremia in schizophrenia or are of potential pharmacogenetic significance. Evidence for functional and/or heterodimerization between wild-type variant orexin receptors and opioid and cannabinoid receptors is discussed in the context of its relevance to depression and epilepsy.

  5. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP receptor expression and function.

    Directory of Open Access Journals (Sweden)

    Anke Bill

    Full Text Available The human prostacyclin receptor (hIP receptor is a seven-transmembrane G protein-coupled receptor (GPCR that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  6. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

    Science.gov (United States)

    Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

    2014-01-01

    The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  7. Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

    Science.gov (United States)

    Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

    2012-04-05

    The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

    International Nuclear Information System (INIS)

    Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

    1989-01-01

    Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

  9. Design and Discovery of Functionally Selective Serotonin 2C (5-HT2C) Receptor Agonists.

    Science.gov (United States)

    Cheng, Jianjun; McCorvy, John D; Giguere, Patrick M; Zhu, Hu; Kenakin, Terry; Roth, Bryan L; Kozikowski, Alan P

    2016-11-10

    On the basis of the structural similarity of our previous 5-HT 2C agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT 2A , 5-HT 2B , and 5-HT 2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT 2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment. The most functionally selective compound (+)-7e produced weak β-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT 2C agonists possessing weak β-arrestin recruitment can produce distinct receptor desensitization properties.

  10. Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Joanna Bandoła

    2017-08-01

    Full Text Available Plasmacytoid dendritic cells (pDCs regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR by the antigen-presenting pDCs, mediated by toll-like receptor (TLR 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

  11. The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

    Science.gov (United States)

    Vainshtein, I; Kovacina, K S; Roth, R A

    2001-03-16

    The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

  12. Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

    Science.gov (United States)

    Bandoła, Joanna; Richter, Cornelia; Ryser, Martin; Jamal, Arshad; Ashton, Michelle P; von Bonin, Malte; Kuhn, Matthias; Dorschner, Benjamin; Alexopoulou, Dimitra; Navratiel, Katrin; Roeder, Ingo; Dahl, Andreas; Hedrich, Christian M; Bonifacio, Ezio; Brenner, Sebastian; Thieme, Sebastian

    2017-01-01

    Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

  13. Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function

    Directory of Open Access Journals (Sweden)

    Alvarez Alejandra R

    2009-11-01

    Full Text Available Abstract Background The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. Results We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. Conclusion Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.

  14. Cloning and functional characterization of three new pheromone receptors from the diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Liu, Yipeng; Liu, Yang; Jiang, Xingchuan; Wang, Guirong

    The highly specialized olfactory receptor neurons (ORNs) on the antennae of male moths can recognize blends of several pheromone components. In previous studies, a total of six candidate pheromone receptor (PR) genes were cloned and functionally characterized in the diamondback moth, Plutella xylostella. In the present work, we report on three novel candidate pheromone receptor genes: PxylOR8, PxylOR41, and PxylOR45 in the same species. Gene expression analysis revealed that PxylOR8 is specifically expressed in female adult antennae, while PxylOR41 and PxylOR45 are expressed in antennae in both sexes, but with a male bias. In situ hybridization revealed that PxylOR8, PxylOR41 and PxylOR45 are localized in long trichoid sensilla. Functional analyses on the three pheromone receptor genes were then performed using the heterologous expression system of Xenopus oocytes. PxylOR41 was tuned to two minor pheromone components Z9-14:Ac, Z9-14:OH, and their analog Z9-14:Ald. PxylOR8 and PxylOR45 did not respond to any tested pheromone components and analogs. These results may contribute to clarifying how pheromone detection works in P. xylostella. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

    Science.gov (United States)

    Miller, Eileen; Czopek, Alicja; Duthie, Karolina M; Kirkby, Nicholas S; van de Putte, Elisabeth E Fransen; Christen, Sibylle; Kimmitt, Robert A; Moorhouse, Rebecca; Castellan, Raphael F P; Kotelevtsev, Yuri V; Kuc, Rhoda E; Davenport, Anthony P; Dhaun, Neeraj; Webb, David J; Hadoke, Patrick W F

    2017-02-01

    The role of smooth muscle endothelin B (ET B ) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ET B receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ET B receptors were selectively deleted from smooth muscle by crossing floxed ET B mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ET B deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ET B was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ET B -mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ET B -mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ET B knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ET B blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ET B -mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ET B knockout mice. In the absence of other pathology, ET B receptors in vascular smooth muscle make a small but significant contribution to ET B -dependent regulation of BP. These ET B receptors have no effect on vascular contraction or neointimal remodeling. © 2016 The Authors.

  16. Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human D1/5 receptors.

    Science.gov (United States)

    Gotzes, F; Balfanz, S; Baumann, A

    1994-01-01

    Members of the superfamily of G-protein coupled receptors share significant similarities in sequence and transmembrane architecture. We have isolated a Drosophila homologue of the mammalian dopamine receptor family using a low stringency hybridization approach. The deduced amino acid sequence is approximately 70% homologous to the human D1/D5 receptors. When expressed in HEK 293 cells, the Drosophila receptor stimulates cAMP production in response to dopamine application. This effect was mimicked by SKF 38393, a specific D1 receptor agonist, but inhibited by dopaminergic antagonists such as butaclamol and flupentixol. In situ hybridization revealed that the Drosophila dopamine receptor is highly expressed in the somata of the optic lobes. This suggests that the receptor might be involved in the processing of visual information and/or visual learning in invertebrates.

  17. IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

    Science.gov (United States)

    Wang, H Y; Paul, W E; Keegan, A D

    1996-02-01

    IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

  18. Neurotransmitters activate T-cells and elicit crucial functions via neurotransmitter receptors.

    Science.gov (United States)

    Levite, Mia

    2008-08-01

    Neurotransmitters are traditionally viewed as nerve-secreted molecules that trigger or inhibit neuronal functions. Yet, neurotransmitters bind also their neurotransmitter receptors in T-cells and directly activate or suppress T-cell functions. This review focuses only on the activating effects of neurotransmitters on T-cells, primarily naïve/resting cells, and covers dopamine, glutamate, serotonin, and few neuropeptides: GnRH-I, GnRH-II, substance P, somatostatin, CGRP, and neuropeptide Y. T-cells express many neurotransmitter receptors. These are regulated by TCR-activation, cytokines, or the neurotransmitters themselves, and are upregulated/downregulated in some human diseases. The context - whether the T-cells are naïve/resting or antigen/mitogen/cytokine-activated, the T-cell subset (CD4/CD8/Th1/Th2/Teff/Treg), neurotransmitter dose (low/optimal or high/excess), exact neurotransmitter receptors expressed, and the cytokine milieu - is crucial, and can determine either activation or suppression of T-cells by the same neurotransmitter. T-cells also produce many neurotransmitters. In summary, neurotransmitters activate vital T-cell functions in a direct, potent and specific manner, and may serve for communicating between the brain and the immune system to elicit an effective and orchestrated immune function, and for new therapeutic avenues, to improve T-cell eradication of cancer and infectious organisms.

  19. Identification of the functional domains of ANT-1, a novel coactivator of the androgen receptor

    International Nuclear Information System (INIS)

    Fan Shuli; Goto, Kiminobu; Chen Guangchun; Morinaga, Hidetaka; Nomura, Masatoshi; Okabe, Taijiro; Nawata, Hajime; Yanase, Toshihiko

    2006-01-01

    Previously, we identified a transcriptional coactivator for the activation function-1 (AF-1) domain of the human androgen receptor (AR) and designated it androgen receptor N-terminal domain transactivating protein-1 (ANT-1). This coactivator, which contains multiple tetratricopeptide repeat (TPR) motifs from amino acid (aa) 294, is identical to a component of U5 small nuclear ribonucleoprotein particles and binds specifically to the AR or glucocorticoid receptor. Here, we identified four distinct functional domains. The AR-AF-1-binding domain, which bound to either aa 180-360 or 360-532 in AR-AF-1, clearly overlapped with TAU-1 and TAU-5. This domain and the subnuclear speckle formation domain in ANT-1 were assigned within the TPR motifs, while the transactivating and nuclear localization signal domains resided within the N-terminal sequence. The existence of these functional domains may further support the idea that ANT-1 can function as an AR-AF-1-specific coactivator while mediating a transcription-splicing coupling

  20. Target-specific M1 inputs to infragranular S1 pyramidal neurons

    Science.gov (United States)

    Fanselow, Erika E.; Simons, Daniel J.

    2016-01-01

    The functional role of input from the primary motor cortex (M1) to primary somatosensory cortex (S1) is unclear; one key to understanding this pathway may lie in elucidating the cell-type specific microcircuits that connect S1 and M1. Recently, we discovered that a subset of pyramidal neurons in the infragranular layers of S1 receive especially strong input from M1 (Kinnischtzke AK, Simons DJ, Fanselow EE. Cereb Cortex 24: 2237–2248, 2014), suggesting that M1 may affect specific classes of pyramidal neurons differently. Here, using combined optogenetic and retrograde labeling approaches in the mouse, we examined the strengths of M1 inputs to five classes of infragranular S1 neurons categorized by their projections to particular cortical and subcortical targets. We found that the magnitude of M1 synaptic input to S1 pyramidal neurons varies greatly depending on the projection target of the postsynaptic neuron. Of the populations examined, M1-projecting corticocortical neurons in L6 received the strongest M1 inputs, whereas ventral posterior medial nucleus-projecting corticothalamic neurons, also located in L6, received the weakest. Each population also possessed distinct intrinsic properties. The results suggest that M1 differentially engages specific classes of S1 projection neurons, thereby regulating the motor-related influence S1 exerts over subcortical structures. PMID:27334960

  1. Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer.

    Science.gov (United States)

    Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Ferré, Sergi

    2014-10-01

    The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. U.S. Government work not protected by U.S. copyright.

  2. Functional Selectivity of Allosteric Interactions within G Protein–Coupled Receptor Oligomers: The Dopamine D1-D3 Receptor Heterotetramer

    Science.gov (United States)

    Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T.; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.

    2014-01-01

    The dopamine D1 receptor–D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa–induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R–D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. PMID:25097189

  3. Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

    Science.gov (United States)

    Norman, Jane E; Cunningham, Margaret R; Jones, Matthew L; Walker, Mary E; Westbury, Sarah K; Sessions, Richard B; Mundell, Stuart J; Mumford, Andrew D

    2016-05-01

    Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists. © 2016 American Heart Association, Inc.

  4. Blockade of cannabinoid CB receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

    DEFF Research Database (Denmark)

    Hansen, H.H.; Ramos, J.A.; Fernández-Ruiz, J.

    2002-01-01

    -induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB receptor function. In contrast, blockade of CB, but not CB, receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest...... receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB/CB receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB and SR144528 for CB) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA...... a critical involvement of CB receptor tonus on neuronal survival following NMDA receptor-induced excitotoxicity in vivo....

  5. Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken

    Directory of Open Access Journals (Sweden)

    Pinheiro Pedro LC

    2012-07-01

    Full Text Available Abstract Background The parathyroid hormone (PTH-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs, which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTH-family members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. Results Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1–34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively. In contrast, PTH-L (100 nM produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively readily activated the receptor. PTHr

  6. CD147/EMMPRIN Acts as a Functional Entry Receptor for Measles Virus on Epithelial Cells▿

    Science.gov (United States)

    Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

    2010-01-01

    Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions. PMID:20147391

  7. CD147/EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells.

    Science.gov (United States)

    Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

    2010-05-01

    Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions.

  8. Neurotransmitter Specific, Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility

    Science.gov (United States)

    Forati, Ebrahim; Sabouni, Abas; Ray, Supriyo; Head, Brian; Schoen, Christian; Sievenpiper, Dan

    2015-01-01

    Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach. PMID:26717196

  9. Vitamin D receptor B1 and exon 1d: functional and evolutionary analysis.

    Science.gov (United States)

    Gardiner, Edith M; Esteban, Luis M; Fong, Colette; Allison, Susan J; Flanagan, Judith L; Kouzmenko, Alexander P; Eisman, John A

    2004-05-01

    The vitamin D receptor (VDR) shares a conserved structural and functional organization with other nuclear receptor (NR) superfamily members. For many NRs, N-terminal variant isoforms that display distinct cell-, stage- and promoter-specific actions have been identified. The novel VDR isoform VDRB1, with a 50 amino acid N-terminal extension, is produced from low abundance transcripts that contain exon 1d of the human VDR locus. There is evidence for the conservation of this exon in other mammalian and avian species. The transactivation differences between VDRB1 and the original VDR, clarified here, provide insights into mechanisms that may contribute to functional differences and potentially distinct physiological roles for these two VDR isoforms.

  10. A functionalized superparamagnetic iron oxide colloid as a receptor directed MR contrast agent

    International Nuclear Information System (INIS)

    Josephson, L.; Groman, E.V.; Menz, E.; Lewis, J.M.; Bengele, H.

    1990-01-01

    We have synthesized a surface functionalized superparamagnetic iron oxide colloid whose clearance from the vascular compartment was inhibited by asialofetuin but not fetuin. Unlike other particulate or colloidal magnetic resonance (MR) contrast agents, the agent of the current communication is not withdrawn from the vascular compartment by cells of the macrophage-monocyte phagocytic system, as indicated by its selective increase in hepatic relaxation rates. Because of this we refer to this colloid as a hepatic selective (HS) MR contrast agent. At 20 mumol Fe/kg the HS MR agent darkened MR images of liver. The HS MR agent exhibited no acute toxicity when injected into rats at 1800 mumol Fe/kg. Based on these observations, surface functionalized superparamagnetic iron oxide colloids may be the basis of MR contrast agents internalized by receptor mediated endocytosis generally, and by the asialoglycoprotein receptor in particular

  11. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    DEFF Research Database (Denmark)

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set...... in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip...... of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact...

  12. Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

    NARCIS (Netherlands)

    Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2010-01-01

    Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

  13. The effect of partial agonist of serotonin-1A receptor on cognitive functions in animal model of schizophrenia

    OpenAIRE

    Antošová, Eliška

    2011-01-01

    Serotoin is a neurotransmitter participating in regulation of many physiologic fuctions. Main serotogenous neurons can be found in nukleus raphe of the brain stem. Nucleus raphe inervates many areas of the brain including the cerebal cortex and hipocampus. These structures are important for controling of higher cognitive functions. 5HT1A receptor is one of many subtypes of serotonin receptors and its activation inhibits iniciating of the action potencials. 5HT1A receptor is expressed presynap...

  14. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

    Science.gov (United States)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    2002-04-01

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

  15. Scavenger receptor AI/II truncation, lung function and COPD: a large population-based study

    DEFF Research Database (Denmark)

    Thomsen, M; Nordestgaard, B G; Tybjærg-Hansen, Anne

    2011-01-01

    The scavenger receptor A-I/II (SRA-I/II) on alveolar macrophages is involved in recognition and clearance of modified lipids and inhaled particulates. A rare variant of the SRA-I/II gene, Arg293X, truncates the distal collagen-like domain, which is essential for ligand recognition. We tested...... whether the Arg293X variant is associated with reduced lung function and risk of chronic obstructive pulmonary disease (COPD) in the general population....

  16. The sigma-1 receptor enhances brain plasticity and functional recovery after experimental stroke

    DEFF Research Database (Denmark)

    Ruscher, Karsten; Shamloo, Mehrdad; Rickhag, Karl Mattias

    2011-01-01

    Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in ...... of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection....

  17. Investigation of serotonin-1A receptor function in the human psychopharmacology of MDMA.

    Science.gov (United States)

    Hasler, F; Studerus, E; Lindner, K; Ludewig, S; Vollenweider, F X

    2009-11-01

    Serotonin (5-HT) release is the primary pharmacological mechanism of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') action in the primate brain. Dopamine release and direct stimulation of dopamine D2 and serotonin 5-HT2A receptors also contributes to the overall action of MDMA. The role of 5-HT1A receptors in the human psychopharmacology of MDMA, however, has not yet been elucidated. In order to reveal the consequences of manipulation at the 5-HT1A receptor system on cognitive and subjective effects of MDMA, a receptor blocking study using the mixed beta-adrenoreceptor blocker/5-HT1A antagonist pindolol was performed. Using a double-blind, placebo-controlled within-subject design, 15 healthy male subjects were examined under placebo (PL), 20 mg pindolol (PIN), MDMA (1.6 mg/kg b.wt.), MDMA following pre-treatment with pindolol (PIN-MDMA). Tasks from the Cambridge Neuropsychological Test Automated Battery were used for the assessment of cognitive performance. Psychometric questionnaires were applied to measure effects of treatment on core dimensions of Altered States of Consciousness, mood and state anxiety. Compared with PL, MDMA significantly impaired sustained attention and visual-spatial memory, but did not affect executive functions. Pre-treatment with PIN did not significantly alter MDMA-induced impairment of cognitive performance and only exerted a minor modulating effect on two psychometric scales affected by MDMA treatment ('positive derealization' and 'dreaminess'). Our findings suggest that MDMA differentially affects higher cognitive functions, but does not support the hypothesis from animal studies, that some of the MDMA effects are causally mediated through action at the 5-HT1A receptor system.

  18. M1 transitions in the (sdg) boson model

    Science.gov (United States)

    Kuyucak, S.; Morrison, I.

    1988-03-01

    Using the {1}/{N} expansion technique we derive expressions for β→g, γ→g and γ→γ M1 transitions in a general boson model. The M1 matrix elements in the sdg-boson model are similar in form to those in the neutron-proton IBM. Comparisons are made to some selected M1 data exhibiting collective character.

  19. Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

    Science.gov (United States)

    Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

    2017-03-01

    Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

    Science.gov (United States)

    O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

    2014-01-01

    Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

  1. Cell model for the study of receptor and regulatory functions of human proHB-EGF

    Directory of Open Access Journals (Sweden)

    N. V. Korotkevych

    2014-08-01

    Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

  2. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    International Nuclear Information System (INIS)

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-01-01

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  3. Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells.

    Science.gov (United States)

    Armstrong, Lynne; Medford, Andrew R L; Uppington, Kay M; Robertson, John; Witherden, Ian R; Tetley, Teresa D; Millar, Ann B

    2004-08-01

    The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase-polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

  4. Elabela-apelin receptor signaling pathway is functional in mammalian systems.

    Science.gov (United States)

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-02-02

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

  5. Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

    Science.gov (United States)

    Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

    2015-01-01

    Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

  6. The Role of Primary Motor Cortex (M1) Glutamate and GABA Signaling in l-DOPA-Induced Dyskinesia in Parkinsonian Rats.

    Science.gov (United States)

    Lindenbach, David; Conti, Melissa M; Ostock, Corinne Y; George, Jessica A; Goldenberg, Adam A; Melikhov-Sosin, Mitchell; Nuss, Emily E; Bishop, Christopher

    2016-09-21

    Long-term treatment of Parkinson's disease with l-DOPA almost always leads to the development of involuntary movements termed l-DOPA-induced dyskinesia. Whereas hyperdopaminergic signaling in the basal ganglia is thought to cause dyskinesia, alterations in primary motor cortex (M1) activity are also prominent during dyskinesia, suggesting that the cortex may represent a therapeutic target. The present study used the rat unilateral 6-hydroxydopamine lesion model of Parkinson's disease to characterize in vivo changes in GABA and glutamate neurotransmission within M1 and determine their contribution to behavioral output. 6-Hydroxydopamine lesion led to parkinsonian motor impairment that was partially reversed by l-DOPA. Among sham-lesioned rats, l-DOPA did not change glutamate or GABA efflux. Likewise, 6-hydroxydopamine lesion did not impact GABA or glutamate among rats chronically treated with saline. However, we observed an interaction of lesion and treatment whereby, among lesioned rats, l-DOPA given acutely (1 d) or chronically (14-16 d) reduced glutamate efflux and enhanced GABA efflux. Site-specific microinjections into M1 demonstrated that l-DOPA-induced dyskinesia was reduced by M1 infusion of a D1 antagonist, an AMPA antagonist, or a GABAA agonist. Overall, the present study demonstrates that l-DOPA-induced dyskinesia is associated with increased M1 inhibition and that exogenously enhancing M1 inhibition may attenuate dyskinesia, findings that are in agreement with functional imaging and transcranial magnetic stimulation studies in human Parkinson's disease patients. Together, our study suggests that increasing M1 inhibitory tone is an endogenous compensatory response designed to limit dyskinesia severity and that potentiating this response is a viable therapeutic strategy. Most Parkinson's disease patients will receive l-DOPA and eventually develop hyperkinetic involuntary movements termed dyskinesia. Such symptoms can be as debilitating as the disease

  7. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  8. Expression and function of β-adrenergic receptors in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Maeki, T.; Andersson, L.C.; Kontula, K.K.

    1992-01-01

    We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

  9. Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

    Directory of Open Access Journals (Sweden)

    Regina T Knapp

    Full Text Available Hsp70 binding protein 1 (HspBP1 and Bcl2-associated athanogene 1 (BAG-1, the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70 chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR, the mineralocorticoid receptor (MR, and the androgen receptor (AR. BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

  10. Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

    NARCIS (Netherlands)

    Ratnala, V.R.; Swarts, H.G.P.; Oostrum, J. van; Leurs, R.; Groot, H.J.M. de; Bakker, R.; Grip, W.J. de

    2004-01-01

    This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera

  11. Resistance to diet-induced adiposity in cannabinoid receptor-1 deficient mice is not due to impaired adipocyte function

    NARCIS (Netherlands)

    Oosterveer, Maaike H.; Koolman, Anniek H.; de Boer, Pieter T.; Bos, Trijnie; Bleeker, Aycha; Bloks, Vincent W.; Kuipers, Folkert; Sauer, Pieter J. J.; van Dijk, Gertjan

    2011-01-01

    Background: Overactivity and/or dysregulation of the endocannabinoid system (ECS) contribute to development of obesity. In vitro studies indicate a regulatory role for the cannabinoid receptor 1 (CB1) in adipocyte function and CB1-receptor deficient (CB1-/-) mice are resistant to high fat

  12. Localization and function of histamine H3 receptor in the nasal mucosa

    OpenAIRE

    Suzuki, Shinya; Takeuchi, Kazuhiko; Majima, Yuichi

    2008-01-01

    BACKGROUND: Histamine is an important chemical mediator of allergic rhinitis (AR). Histamine H3 receptors H3R are located on cholinergic and NANC neurons of the myenteric plexus, and activation of H3R regulates gastric acid secretion. However, little is known about the localization and function of H3R in the upper airway. OBJECTIVE: The objective of this study was to examine the localization and possible function of H3R in the nasal mucosa. METHODS: We extracted total RNA from the inferior tu...

  13. Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

    Science.gov (United States)

    Cook, Donald N.; Kang, Hong Soon; Jetten, Anton M.

    2015-01-01

    In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs). We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated. PMID:26878025

  14. Retinoic Acid-Related Orphan Receptors (RORs: Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

    Directory of Open Access Journals (Sweden)

    Donald N. Cook

    2015-12-01

    Full Text Available In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs. We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated.

  15. An energetic orphan in an endocrine tissue: a revised perspective of the function of estrogen receptor-related receptor alpha in bone and cartilage.

    Science.gov (United States)

    Bonnelye, Edith; Aubin, Jane E

    2013-02-01

    Estrogen receptor-related receptor alpha (ERRα) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ERα/β, but it does not bind estrogen. ERRα not only plays a functional role in osteoblasts but also in osteoclasts and chondrocytes. In addition, the ERRs, including ERRα, can be activated by coactivators such as peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC1α and β) and are implicated in adipogenesis, fatty acid oxidation, and oxidative stress defense, suggesting that ERRα-through its activity in bone resorption and adipogenesis--may regulate the insulin and leptin pathways and contribute to aging-related changes in bone and cartilage. In this review, we discuss data on ERRα and its cellular and molecular modes of action, which have broad implications for considering the potential role of this orphan receptor in cartilage and bone endocrine function, on whole-organism physiology, and in the bone aging process. Copyright © 2013 American Society for Bone and Mineral Research.

  16. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Dieudonnée Togbe

    Full Text Available BACKGROUND: TNF-related lymphotoxin alpha (LTalpha is essential for the development of Plasmodium berghei ANKA (PbA-induced experimental cerebral malaria (ECM. The pathway involved has been attributed to TNFR2. Here we show a second arm of LTalpha-signaling essential for ECM development through LTbeta-R, receptor of LTalpha1beta2 heterotrimer. METHODOLOGY/PRINCIPAL FINDINGS: LTbetaR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTalphabeta deficient mice. Resistance of LTalphabeta or LTbetaR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin(+ CD8(+ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTbetaR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM. CONCLUSIONS/SIGNIFICANCE: LTbetaR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTbetaR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.

  17. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    International Nuclear Information System (INIS)

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  18. Cloning and functional characterization of the rabbit C-C chemokine receptor 2

    Directory of Open Access Journals (Sweden)

    Hamdouchi Chafiq

    2005-07-01

    Full Text Available Abstract Background CC-family chemokine receptor 2 (CCR2 is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model. Results Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1 with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1α or MIP-1β. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis. Conclusion In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1.

  19. Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

    Science.gov (United States)

    DeBell, Karen E; Simhadri, Venkateswara R; Mariano, John L; Borrego, Francisco

    2012-04-26

    Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes. We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity. These studies provide new insights into the function of CD300a in tuning T and B cell responses.

  20. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    International Nuclear Information System (INIS)

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-01-01

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  1. Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

    Science.gov (United States)

    Hasenkamp, Sandra; Telgmann, Ralph; Staessen, Jan A; Hagedorn, Claudia; Dördelmann, Corinna; Bek, Martin; Brand-Herrmann, Stefan-Martin; Brand, Eva

    2008-10-01

    The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

  2. Impact of blood processing variations on Natural Killer cell frequency, activation, chemokine receptor expression and function

    Science.gov (United States)

    Naranbhai, Vivek; Bartman, Pat; Ndlovu, Dudu; Ramkalawon, Pamela; Ndung’u, Thumbi; Wilson, Douglas; Altfeld, Marcus; Carr, William H

    2011-01-01

    Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 hrs) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated NK cells and modified chemokine receptor expression. Delays in processing beyond 8 hours activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies. PMID:21255578

  3. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Science.gov (United States)

    2012-01-01

    Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

  4. Multiple functions and essential roles of nuclear receptor coactivators of bHLH-PAS family.

    Science.gov (United States)

    Pecenova, L; Farkas, Robert

    2016-07-01

    Classical non-peptide hormones, such as steroids, retinoids, thyroid hormones, vitamin D3 and their derivatives including prostaglandins, benzoates, oxysterols, and bile acids, are collectively designated as small lipophilic ligands, acting via binding to the nuclear receptors (NRs). The NRs form a large superfamily of transcription factors that participate virtually in every key biological process. They control various aspects of animal development, fertility, gametogenesis, and numerous metabolic pathways, and can be misregulated in many types of cancers. Their enormous functional plasticity, as transcription factors, relates in part to NR-mediated interactions with plethora of coregulatory proteins upon ligand binding to their ligand binding domains (LBD), or following covalent modification. Here, we review some general views of a specific group of NR coregulators, so-called nuclear receptor coactivators (NRCs) or steroid receptor coactivators (SRCs) and highlight some of their unique functions/roles, which are less extensively mentioned and discussed in other reviews. We also try to pinpoint few neglected moments in the cooperative action of SRCs, which may also indicate their variable roles in the hormone-independent signaling pathways.

  5. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

    Science.gov (United States)

    Parrish-Novak, J; Dillon, S R; Nelson, A; Hammond, A; Sprecher, C; Gross, J A; Johnston, J; Madden, K; Xu, W; West, J; Schrader, S; Burkhead, S; Heipel, M; Brandt, C; Kuijper, J L; Kramer, J; Conklin, D; Presnell, S R; Berry, J; Shiota, F; Bort, S; Hambly, K; Mudri, S; Clegg, C; Moore, M; Grant, F J; Lofton-Day, C; Gilbert, T; Rayond, F; Ching, A; Yao, L; Smith, D; Webster, P; Whitmore, T; Maurer, M; Kaushansky, K; Holly, R D; Foster, D

    2000-11-02

    Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

  6. Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

    Science.gov (United States)

    Yamada, M; Yamada, M; Lombet, A; Forgez, P; Rostène, W

    1998-01-01

    Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.

  7. Corepressive function of nuclear receptor coactivator 2 in androgen receptor of prostate cancer cells treated with antiandrogen

    International Nuclear Information System (INIS)

    Takeda, Keisuke; Hara, Noboru; Nishiyama, Tsutomu; Tasaki, Masayuki; Ishizaki, Fumio; Tomita, Yoshihiko

    2016-01-01

    Recruitment of cofactors in the interaction of the androgen receptor (AR) and AR ligands plays a critical role in determining androgenic/antiandrogenic effects of the AR ligand on signaling, but the functions of key cofactors, including nuclear receptor coactivator (NCOA), remain poorly understood in prostate cancer cells treated with AR ligands. We examined prostate cancer cell lines LNCaP and VCaP expressing mutated and wild-type ARs, respectively, to clarify the significance of NCOAs in the effect of antiandrogens. Hydroxyflutamide showed antagonistic activity against VCaP and an agonistic effect on LNCaP. Bicalutamide served as an antagonist for both. We analyzed mRNA transcription and protein expression of NCOAs in these cells pretreated with dihydrotestosterone and thereafter treated with the mentioned antiandrogens. Transcriptional silencing of candidate NCOAs and AR was performed using small interfering RNA (siRNA). Cell proliferation was evaluated with MTT assay. LNCaP treated with bicalutamide showed an about four-fold increase in the expression of NCOA2 mRNA compared to those pretreated with dihydrotestosterone alone (P <0.01). In VCaP pretreated with dihydrotestosterone, transcriptions of NCOA2 and NCOA7 were slightly increased with bicalutamide (1.96- and 2.42-fold, respectively) and hydroxyflutamide (1.33-fold in both). With Western blotting, the expression of NCOA2 protein also increased in LNCaP cells treated with bicalutamide compared with that in control cells pretreated with dihydrotestosterone alone. Following silencing with siRNA for NCOA2, PSA levels in media with LNCaP receiving bicalutamide were elevated compared with those in non-silencing controls (101.6 ± 4.2 vs. 87.8 ± 1.4 ng/mL, respectively, P =0.0495). In LNCaP cells treated with dihydrotestosterone and bicalutamide, NCOA2-silencing was associated with a higher proliferation activity compared with non-silencing control and AR-silencing. NCOA2, which has been thought to be recruited

  8. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Directory of Open Access Journals (Sweden)

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  9. The FKBP51 Glucocorticoid Receptor Co-Chaperone: Regulation, Function, and Implications in Health and Disease.

    Science.gov (United States)

    Fries, Gabriel R; Gassen, Nils C; Rein, Theo

    2017-12-05

    Among the chaperones and co-chaperones regulating the glucocorticoid receptor (GR), FK506 binding protein (FKBP) 51 is the most intensely investigated across different disciplines. This review provides an update on the role of the different co-chaperones of Hsp70 and Hsp90 in the regulation of GR function. The development leading to the focus on FKBP51 is outlined. Further, a survey of the vast literature on the mechanism and function of FKBP51 is provided. This includes its structure and biochemical function, its regulation on different levels-transcription, post-transcription, and post-translation-and its function in signaling pathways. The evidence portraying FKBP51 as a scaffolding protein organizing protein complexes rather than a chaperone contributing to the folding of individual proteins is collated. Finally, FKBP51's involvement in physiology and disease is outlined, and the promising efforts in developing drugs targeting FKBP51 are discussed.

  10. Serotonin 1B Receptors Regulate Prefrontal Function by Gating Callosal and Hippocampal Inputs

    DEFF Research Database (Denmark)

    Kjaerby, Celia; Athilingam, Jegath; Robinson, Sarah E

    2016-01-01

    Both medial prefrontal cortex (mPFC) and serotonin play key roles in anxiety; however, specific mechanisms through which serotonin might act on the mPFC to modulate anxiety-related behavior remain unknown. Here, we use a combination of optogenetics and synaptic physiology to show that serotonin...... acts presynaptically via 5-HT1B receptors to selectively suppress inputs from the contralateral mPFC and ventral hippocampus (vHPC), while sparing those from mediodorsal thalamus. To elucidate how these actions could potentially regulate prefrontal circuit function, we infused a 5-HT1B agonist...... into the mPFC of freely behaving mice. Consistent with previous studies that have optogenetically inhibited vHPC-mPFC projections, activating prefrontal 5-HT1B receptors suppressed theta-frequency mPFC activity (4-12 Hz), and reduced avoidance of anxiogenic regions in the elevated plus maze. These findings...

  11. HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis

    Science.gov (United States)

    Harrington, Willie; Bond, Vincent; Huang, Ming Bo; Powell, Michael; Lillard, James; Manne, Upender; Bumpers, Harvey

    2010-01-01

    CXCR4 receptors have been implicated in tumorigenesis and proliferation, making it a potential target for colorectal cancer therapy. Expression of this chemokine receptor on cellular surfaces appears to promote metastasis by directly stimulating tumor cell migration and invasion. The receptor/ligand, CXCR4/SDF-1α, pair are critically important to angiogenesis and vascular remodeling which supports cancer proliferation. Our work has shown that a novel apoptotic peptide of HIV-1, Nef-M1, can act as a CXCR4 antagonist, inducing apoptosis in CXCR4 containing cells. Four colorectal tumor cell lines (HT-29, LS174t, SW480, WiDr), were evaluated for their response to Nef-M1 peptide via in vivo and in vitro. The presence of CXCR4 receptors on tumor cells was determined using immunohistochemical and RT-PCR analyses. Solid xenografts derived from tumor cell lines grown in SCID mice, were evaluated for the persistence of the receptor. Xenografts propagated in SCID mice from each of the four cell lines demonstrated high levels of receptor expression as well. The effects of Nef-M1 in vivo via splenic injected mice and subsequent hepatic metastasis also demonstrated dramatic reduction of primary tumor growth in the spleen and secondary invasion of the liver. We concluded that Nef-M1 peptide, through physical interaction(s) with CXCR4, drives apoptotic reduction in in vivo primary tumor growth and metastasis. PMID:20383296

  12. Thyroid hormone and retinoid X receptor function and expression during sea lamprey (Petromyzon marinus) metamorphosis.

    Science.gov (United States)

    Manzon, Lori A; Youson, John H; Holzer, Guillaume; Staiano, Leopoldo; Laudet, Vincent; Manzon, Richard G

    2014-08-01

    Sea lampreys (Petromyzon marinus) are members of the ancient class Agnatha and undergo a metamorphosis that transforms blind, sedentary, filter-feeding larvae into free-swimming, parasitic juveniles. Thyroid hormones (THs) appear to be important for lamprey metamorphosis, however, serum TH concentrations are elevated in the larval phase, decline rapidly during early metamorphosis and remain low until metamorphosis is complete; these TH fluctuations are contrary to those of other metamorphosing vertebrates. Moreover, thyroid hormone synthesis inhibitors (goitrogens) induce precocious metamorphosis and exogenous TH treatments disrupt natural metamorphosis in P. marinus. Given that THs exert their effects by binding to TH nuclear receptors (TRs) that often act as heterodimers with retinoid X receptors (RXRs), we cloned and characterized these receptors from P. marinus and examined their expression during metamorphosis. Two TRs (PmTR1 and PmTR2) and three RXRs (PmRXRs) were isolated from P. marinus cDNA. Phylogenetic analyses group the PmTRs together on a branch prior to the gnathostome TRα/β split. The three RXRs also group together, but our data indicated that these transcripts are most likely either allelic variants of the same gene locus, or the products of a lamprey-specific duplication event. Importantly, these P. marinus receptors more closely resemble vertebrate as opposed to invertebrate chordate receptors. Functional analysis revealed that PmTR1 and PmTR2 can activate transcription of TH-responsive genes when treated with nanomolar concentrations of TH and they have distinct pharmacological profiles reminiscent of vertebrate TRβ and TRα, respectively. Also similar to other metamorphosing vertebrates, expression patterns of the PmTRs during lamprey metamorphosis suggest that PmTR1 has a dynamic, tissue-specific expression pattern that correlates with tissue morphogenesis and biochemical changes and PmTR2 has a more uniform expression pattern. This TR

  13. The effect of proton correlations in the M1 scattering strengths of even calcium isotopes

    International Nuclear Information System (INIS)

    Chaves, L.; Poves, A.

    1986-01-01

    The proton correlations are evaluated in Ca isotopes using the wave functions obtained in an extended shell model calculation in the [1dsub(3/2), 1fsub(7/2), 1fsub(5/2)] valence space. The inclusion of 2p-2h correlations in M1 scattering strengths and magnetic moments improves the agrreement with the experimental data. The M1 strength in 40 Ca is correctly reproduced. (orig.)

  14. Muscarinic receptor subtype mRNA expression in the human prostate: association with age, pathological diagnosis, prostate size, or potentially interfering medications?

    NARCIS (Netherlands)

    Witte, Lambertus P. W.; Teitsma, Christine A.; de La Rosette, Jean J. M. C. H.; Michel, Martin C.

    2014-01-01

    As the prostate abundantly expresses muscarinic receptors and antagonists for such receptors are increasingly used in the treatment of men with voiding function and large prostates, we have explored an association of the mRNA expression of human M1, M2, M3, M4, and M5 receptors in human prostate

  15. Gonadotropins, their receptors, and the regulation of testicular functions in fish

    NARCIS (Netherlands)

    Schulz, Rüdiger W; Vischer, H F; Cavaco, J E; Dos Santos Rocha, M.E.; Tyler, R.C.; Goos, H.J.; Bogerd, J.

    The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the

  16. β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Science.gov (United States)

    Lamkin, Donald M.; Ho, Hsin-Yun; Ong, Tiffany H.; Kawanishi, Carly K.; Stoffers, Victoria L.; Ahlawat, Nivedita; Ma, Jeffrey C.Y.; Arevalo, Jesusa M. G.; Cole, Steve W.; Sloan, Erica K.

    2016-01-01

    β-adrenergic signaling can regulate macrophage involvement in several diseases and often produces anti-inflammatory properties in macrophages, which are similar to M2 properties in a dichotomous M1 vs. M2 macrophage taxonomy. However, it is not clear that β-adrenergic-stimulated macrophages may be classified strictly as M2. In this in vitro study, we utilized recently published criteria and transcriptome-wide bioinformatics methods to map the relative polarity of murine β-adrenergic-stimulated macrophages within a wider M1–M2 spectrum. Results show that β-adrenergic-stimulated macrophages did not fit entirely into any one predefined category of the M1–M2 spectrum but did express genes that are representative of some M2 side categories. Moreover, transcript origin analysis of genome-wide transcriptional profiles located β-adrenergic-stimulated macrophages firmly on the M2 side of the M1–M2 spectrum and found active suppression of M1 side gene transcripts. The signal transduction pathways involved were mapped through blocking experiments and bioinformatics analysis of transcription factor binding motifs. M2-promoting effects were mediated specifically through β2-adrenergic receptors and were associated with CREB, C/EBPβ, and ATF transcription factor pathways but not with established M1–M2 STAT pathways. Thus, β-adrenergic-signaling induces a macrophage transcriptome that locates on the M2 side of the M1–M2 spectrum but likely accomplishes this effect through a signaling pathway that is atypical for M2-spectrum macrophages. PMID:27485040

  17. Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function

    International Nuclear Information System (INIS)

    Nagayama, Yuji; Wadsworth, H.L.; Chazenbalk, G.D.; Russo, D.; Seto, Pui; Rapoport, B.

    1991-01-01

    To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction the authors constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites

  18. Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

    Science.gov (United States)

    Le, N; Simon, M A

    1998-08-01

    DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

  19. Loss of melanocortin-4 receptor function attenuates HPA responses to psychological stress

    DEFF Research Database (Denmark)

    Ryan, Karen K; Mul, Joram D; Clemmensen, Christoffer

    2014-01-01

    function. These results support the hypothesis that endogenous MC4R signaling contributes to the HPA axis response to stress. Because MC4R plays a critical role in the regulation of energy balance, the present work suggests that it may also serve as an important communication link between brain metabolic...... in hypothalamic-pituitary-adrenocortical axis (HPA) regulation. The present work investigated the role of chronic Mc4r function to modulate basal HPA axis tone and to facilitate acute HPA responses to psychological stress, using a novel rat model with Mc4r loss-of-function. In this study, adult male rats were......The melanocortin 4 receptor (MC4R), well-known for its role in the regulation of energy balance, is widely expressed in stress-regulatory brain regions, including the paraventricular nucleus of the hypothalamus (PVH) and the medial amygdala (MeA). In agreement with this, MC4R has been implicated...

  20. Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors.

    Science.gov (United States)

    Chen, Min; Georgeson, Keith E; Harmon, Carroll M; Haskell-Luevano, Carrie; Yang, Yingkui

    2006-11-01

    The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.

  1. Spatiotemporal intracellular dynamics of neurotrophin and its receptors. Implications for neurotrophin signaling and neuronal function.

    Science.gov (United States)

    Bronfman, F C; Lazo, O M; Flores, C; Escudero, C A

    2014-01-01

    Neurons possess a polarized morphology specialized to contribute to neuronal networks, and this morphology imposes an important challenge for neuronal signaling and communication. The physiology of the network is regulated by neurotrophic factors that are secreted in an activity-dependent manner modulating neuronal connectivity. Neurotrophins are a well-known family of neurotrophic factors that, together with their cognate receptors, the Trks and the p75 neurotrophin receptor, regulate neuronal plasticity and survival and determine the neuronal phenotype in healthy and regenerating neurons. Is it now becoming clear that neurotrophin signaling and vesicular transport are coordinated to modify neuronal function because disturbances of vesicular transport mechanisms lead to disturbed neurotrophin signaling and to diseases of the nervous system. This chapter summarizes our current understanding of how the regulated secretion of neurotrophin, the distribution of neurotrophin receptors in different locations of neurons, and the intracellular transport of neurotrophin-induced signaling in distal processes are achieved to allow coordinated neurotrophin signaling in the cell body and axons.

  2. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  3. P2X7 Receptor Function in Bone-Related Cancer

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    Elena Adinolfi

    2012-01-01

    Full Text Available Modulation of tumor microenvironment by different mediators is central in determining neoplastic formation and progression. Among these molecules extracellular ATP is emerging as a good candidate in promoting cell growth, neovascularization, tumor-host interactions, and metastatization. This paper summarizes recent findings on expression and function of P2X7 receptor for extracellular ATP in primary and metastatic bone cancers. Search of mRNA expression microchip databases and literature analysis demonstrate a high expression of P2X7 in primary bone tumors as well as in other malignancies such as multiple myeloma, neuroblastoma, breast, and prostate cancer. Evidence that P2X7 triggers NFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting either primary tumor development or osteoblastic lesions is also reported. Moreover, P2X7 receptor is involved in osteoclast differentiation, RANKL expression, matrix metalloproteases and cathepsin secretion thus promoting bone resorption and osteolytic lesions. Taken together these data point to a pivotal role for the P2X7 receptor in bone cancer biology.

  4. Dibutyltin disrupts glucocorticoid receptor function and impairs glucocorticoid-induced suppression of cytokine production.

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    Christel Gumy

    Full Text Available BACKGROUND: Organotins are highly toxic and widely distributed environmental chemicals. Dibutyltin (DBT is used as stabilizer in the production of polyvinyl chloride plastics, and it is also the major metabolite formed from tributyltin (TBT in vivo. DBT is immunotoxic, however, the responsible targets remain to be defined. Due to the importance of glucocorticoids in immune-modulation, we investigated whether DBT could interfere with glucocorticoid receptor (GR function. METHODOLOGY: We used HEK-293 cells transiently transfected with human GR as well as rat H4IIE hepatoma cells and native human macrophages and human THP-1 macrophages expressing endogenous receptor to study organotin effects on GR function. Docking of organotins was used to investigate the binding mechanism. PRINCIPAL FINDINGS: We found that nanomolar concentrations of DBT, but not other organotins tested, inhibit ligand binding to GR and its transcriptional activity. Docking analysis indicated that DBT inhibits GR activation allosterically by inserting into a site close to the steroid-binding pocket, which disrupts a key interaction between the A-ring of the glucocorticoid and the GR. DBT inhibited glucocorticoid-induced expression of phosphoenolpyruvate carboxykinase (PEPCK and tyrosine-aminotransferase (TAT and abolished the glucocorticoid-mediated transrepression of TNF-alpha-induced NF-kappaB activity. Moreover, DBT abrogated the glucocorticoid-mediated suppression of interleukin-6 (IL-6 and TNF-alpha production in lipopolysaccharide (LPS-stimulated native human macrophages and human THP-1 macrophages. CONCLUSIONS: DBT inhibits ligand binding to GR and subsequent activation of the receptor. By blocking GR activation, DBT may disturb metabolic functions and modulation of the immune system, providing an explanation for some of the toxic effects of this organotin.

  5. Proteinase-Activated Receptor 1 (PAR1 regulates leukemic stem cell functions.

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    Nicole Bäumer

    Full Text Available External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

  6. Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

    Science.gov (United States)

    Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

    2017-01-15

    Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Distinct functions and regulation of epithelial progesterone receptor in the mouse cervix, vagina, and uterus.

    Science.gov (United States)

    Mehta, Fabiola F; Son, Jieun; Hewitt, Sylvia C; Jang, Eunjung; Lydon, John P; Korach, Kenneth S; Chung, Sang-Hyuk

    2016-04-05

    While the function of progesterone receptor (PR) has been studied in the mouse vagina and uterus, its regulation and function in the cervix has not been described. We selectively deleted epithelial PR in the female reproductive tracts using the Cre/LoxP recombination system. We found that epithelial PR was required for induction of apoptosis and suppression of cell proliferation by progesterone (P4) in the cervical and vaginal epithelium. We also found that epithelial PR was dispensable for P4 to suppress apoptosis and proliferation in the uterine epithelium. PR is encoded by the Pgr gene, which is regulated by estrogen receptor α (ERα) in the female reproductive tracts. Using knock-in mouse models expressing ERα mutants, we determined that the DNA-binding domain (DBD) and AF2 domain of ERα were required for upregulation of Pgr in the cervix and vagina as well as the uterine stroma. The ERα AF1 domain was required for upregulation of Pgr in the vaginal stroma and epithelium and cervical epithelium, but not in the uterine and cervical stroma. ERα DBD, AF1, and AF2 were required for suppression of Pgr in the uterine epithelium, which was mediated by stromal ERα. Epithelial ERα was responsible for upregulation of epithelial Pgr in the cervix and vagina. Our results indicate that regulation and functions of epithelial PR are different in the cervix, vagina, and uterus.

  8. Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.

    Science.gov (United States)

    Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E; Müller-Tidow, Carsten; Tickenbrock, Lara

    2014-01-01

    External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

  9. Testosterone receptor blockade after trauma-hemorrhage improves cardiac and hepatic functions in males.

    Science.gov (United States)

    Remmers, D E; Wang, P; Cioffi, W G; Bland, K I; Chaudry, I H

    1997-12-01

    Although studies have shown that testosterone receptor blockade with flutamide after hemorrhage restores the depressed immune function, it remains unknown whether administration of flutamide following trauma and hemorrhage and resuscitation has any salutary effects on the depressed cardiovascular and hepatocellular functions. To study this, male rats underwent a laparotomy (representing trauma) and were then bled and maintained at a mean arterial pressure (MAP) of 40 mmHg until the animals could not maintain this pressure. Ringer lactate was given to maintain a MAP of 40 mmHg until 40% of the maximal shed blood volume was returned in the form of Ringer lactate. The rats were then resuscitated with four times the shed blood volume in the form of Ringer lactate over 60 min. Flutamide (25 mg/kg) or an equal volume of the vehicle propanediol was injected subcutaneously 15 min before the end of resuscitation. Various in vivo heart performance parameters (e.g., maximal rate of the pressure increase or decrease), cardiac output, and hepatocellular function (i.e., the maximum velocity and the overall efficiency of indocyanine green clearance) were determined at 20 h after resuscitation. Additionally, hepatic microvascular blood flow (HMBF) was determined using a laser Doppler flowmeter. The results indicate that left ventricular performance, cardiac output, HMBF, and hepatocellular function decreased significantly at 20 h after the completion of trauma, hemorrhage, and resuscitation. Administration of the testosterone receptor blocker flutamide, however, significantly improved cardiac performance, HMBF, and hepatocellular function. Thus flutamide appears to be a novel and useful adjunct for improving cardiovascular and hepatocellular functions in males following trauma and hemorrhagic shock.

  10. Repression of estrogen receptor β function by putative tumor suppressor DBC1

    International Nuclear Information System (INIS)

    Koyama, Satoshi; Wada-Hiraike, Osamu; Nakagawa, Shunsuke; Tanikawa, Michihiro; Hiraike, Haruko; Miyamoto, Yuichiro; Sone, Kenbun; Oda, Katsutoshi; Fukuhara, Hiroshi; Nakagawa, Keiichi; Kato, Shigeaki; Yano, Tetsu; Taketani, Yuji

    2010-01-01

    It has been well established that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor (ER) α appears to promote the proliferation of cancer tissues, while ERβ can protect against the mitogenic effect of estrogen in breast tissue. The expression status of ERα and ERβ may greatly influence on the development, treatment, and prognosis of breast cancer. Previous studies have indicated that the deleted in breast cancer 1 (DBC1/KIAA1967) gene product has roles in regulating functions of nuclear receptors. The gene encoding DBC1 is a candidate for tumor suppressor identified by genetic search for breast cancer. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of the endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. In addition, DBC1 modulates ERα expression and promotes breast cancer cell survival by binding to ERα. Here we report an ERβ-specific repressive function of DBC1. Immunoprecipitation and immunofluorescence studies show that ERβ and DBC1 interact in a ligand-independent manner similar to ERα. In vitro pull-down assays revealed a direct interaction between DBC1 amino-terminus and activation function-1/2 domain of ERβ. Although DBC1 shows no influence on the ligand-dependent transcriptional activation function of ERα, the expression of DBC1 negatively regulates the ligand-dependent transcriptional activation function of ERβin vivo, and RNA interference-mediated depletion of DBC1 stimulates the transactivation function of ERβ. These results implicate the principal role of DBC1 in regulating ERβ-dependent gene expressions.

  11. Symbiotic nature of the object M1-77

    International Nuclear Information System (INIS)

    Kondrt'eva, L.N.

    2004-01-01

    Many year spectral observations show, that the object M1-77 is the symbiotic system, which consists of a M-giant and a B-star. An emission spectra arises from an envelope, which was formed from a giant's extended atmosphere, and now is ionized by the hotter component. Some spectral changes were registered in M1-77: the forbidden lines intensities increase relatively to that of Hα. It is connected with the decrease of hydrogen emission. (author)

  12. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

    OpenAIRE

    Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

    2010-01-01

    Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the pre...

  13. Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct.

    Science.gov (United States)

    Orihuela, Pedro A; Zuñiga, Lidia M; Rios, Mariana; Parada-Bustamante, Alexis; Sierralta, Walter D; Velásquez, Luis A; Croxatto, Horacio B

    2009-11-30

    Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

  14. A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

    Science.gov (United States)

    Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

    2014-05-01

    The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

  15. Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    2017-08-01

    Full Text Available Natural-killer receptor group 2, member D (NKG2D is a well characterized natural killer (NK cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs, DNAX accessory molecule-1 (DNAM1 and CD16, will be also discussed.

  16. Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

    Science.gov (United States)

    Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

    2017-07-01

    Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

  17. Functional expression of ionotropic purinergic receptors on mouse taste bud cells.

    Science.gov (United States)

    Hayato, Ryotaro; Ohtubo, Yoshitaka; Yoshii, Kiyonori

    2007-10-15

    Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

  18. Nicotinic receptors and functional regulation of GABA cell microcircuitry in bipolar disorder and schizophrenia.

    Science.gov (United States)

    Benes, Francine M

    2012-01-01

    Studies of the hippocampus in postmortem brains from patients with schizophrenia and bipolar disorder have provided evidence for a defect of GABAergic interneurons. Significant decreases in the expression of GAD67, a marker for GABA cell function, have been found repeatedly in several different brain regions that include the hippocampus. In this region, nicotinic receptors are thought to play an important role in modulating the activity of GABAergic interneurons by influences of excitatory cholinergic afferents on their activity. In bipolar disorder, this influence appears to be particularly prominent in the stratum oriens of sectors CA3/2 and CA1, two sites where these cells constitute the exclusive neuronal cell type. In sector CA3/2, this layer receives a robust excitatory projection from the basolateral amygdala (BLA) and this is thought to play a central role in regulating GABA cells at this locus. Using laser microdissection, recent studies have focused selectively on these two layers and their associated GABA cells using microarray technology. The results have provided support for the idea that nicotinic cholinergic receptors play a particularly important role in regulating the activity of GABA neurons at these loci by regulating the progression of cell cycle and the repair of damaged DNA. In bipolar disorder, there is a prominent reduction in the expression of mRNAs for several different nicotinic subunit isoforms. These decreases could reflect a diminished influence of this receptor system on these GABA cells, particularly in sector CA3/2 where a preponderance of abnormalities have been observed in postmortem studies. In patients with bipolar disorder, excitatory nicotinic cholinergic fibers from the medial septum may converge with glutamatergic fibers from the BLA on GABAergic interneurons in the stratum oriens of CA3/2 and result in disturbances of their genomic and functional integrity, ones that may induce disruptions of the integration of

  19. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

    Science.gov (United States)

    Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

    2010-01-01

    Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-κB ligand in the presence or absence of the A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A1R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A1R antagonist DPCPX inhibits osteoclast formation (IC50=1 nM), with altered morphology and reduced ability to resorb bone. A1R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A1R and further suggest that A1R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.—Kara, F. M., Chitu, V., Sloane, J., Axelrod, M., Fredholm, B. B., Stanley, R., Cronstein, B. N. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. PMID:20181934

  20. Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

    International Nuclear Information System (INIS)

    Lukas, R.J.; Cullen, M.J.

    1988-01-01

    An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors

  1. Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

    International Nuclear Information System (INIS)

    Kohno, Kenji; Hayes, H.; Mekada, Eisuke; Uchida, Tsuyoshi

    1987-01-01

    A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. 125 I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH 4 Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1,000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells

  2. Role of type I interferon receptor signaling on NK cell development and functions.

    Directory of Open Access Journals (Sweden)

    Jean Guan

    Full Text Available Type I interferons (IFN are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

  3. A comparative review of Toll-like receptor 4 expression and functionality in different animal species

    Directory of Open Access Journals (Sweden)

    Céline eVAURE

    2014-07-01

    Full Text Available Toll-like receptors (TLRs belong to the pattern recognition receptor (PRR family, a key component of the innate immune system. TLRs detect invading pathogens and initiate an immediate immune response to them, followed by a long-lasting adaptive immune response. Activation of TLRs leads to the synthesis of pro-inflammatory cytokines and chemokines and the expression of co-stimulatory molecules. TLR4 specifically recognizes bacterial lipopolysaccharide (LPS, along with several other components of pathogens and endogenous molecules produced during abnormal situations, such as tissue damage. Evolution across species can lead to substantial diversity in the TLR4’s affinity and specificity to its ligands, the TLR4 gene and cellular expression patterns and tissue distribution. Consequently, TLR4 functions vary across different species. In recent years, the use of synthetic TLR agonists as adjuvants has emerged as a realistic therapeutic goal, notably for the development of vaccines against poorly immunogenic targets. Given that an adjuvanted vaccine must be assessed in pre-clinical animal models before being tested in humans, the extent to which an animal model represents and predicts the human condition is of particular importance. This review focuses on the current knowledge on the critical points of divergence between human and the mammalian species commonly used in vaccine research and development (non-human primate, mouse, rat, rabbit, swine and dog, in terms of molecular, cellular and functional properties of TLR4.

  4. Kidins220/ARMS as a functional mediator of multiple receptor signalling pathways.

    Science.gov (United States)

    Neubrand, Veronika E; Cesca, Fabrizia; Benfenati, Fabio; Schiavo, Giampietro

    2012-04-15

    An increasing body of evidence suggests that several membrane receptors--in addition to activating distinct signalling cascades--also engage in substantial crosstalk with each other, thereby adjusting their signalling outcome as a function of specific input information. However, little is known about the molecular mechanisms that control their coordination and integration of downstream signalling. A protein that is likely to have a role in this process is kinase-D-interacting substrate of 220 kDa [Kidins220, also known as ankyrin repeat-rich membrane spanning (ARMS), hereafter referred to as Kidins220/ARMS]. Kidins220/ARMS is a conserved membrane protein that is preferentially expressed in the nervous system and interacts with the microtubule and actin cytoskeleton. It interacts with neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, and is a common downstream target of several trophic stimuli. Kidins220/ARMS is required for neuronal differentiation and survival, and its expression levels modulate synaptic plasticity. Kidins220/ARMS knockout mice show developmental defects mainly in the nervous and cardiovascular systems, suggesting a crucial role for this protein in modulating the cross talk between different signalling pathways. In this Commentary, we summarise existing knowledge regarding the physiological functions of Kidins220/ARMS, and highlight some interesting directions for future studies on the role of this protein in health and disease.

  5. Augmented macrophage differentiation and polarization of tumor-associated macrophages towards M1 subtype in listeria-administered tumor-bearing host.

    Science.gov (United States)

    Rai, Rakesh K; Vishvakarma, Naveen K; Mohapatra, Tribhuban M; Singh, Sukh Mahendra

    2012-09-01

    This study investigates the effect of Listeria administration on differentiation of macrophages from precursor bone marrow cells and functional status of tumor-associated macrophages (TAM). Listeria administration not only resulted in an augmented infiltration of tumor by F4/80 macrophages but also repolarized the functional status of TAM displaying features of some M1 macrophage subtype with upregulated phagocytosis and tumoricidal activity accompanied by altered expression of monocarboxylate transporter-1, toll-like receptor-2, surface markers: CD11c, interleukin-2 receptor, CD62L, and secreted molecules: nitric oxide, interleukin (IL)-1, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor. Declined tumor cell survival and modulated repertoire of cytokines: interferon-γ, IL-6, IL-10, and transforming growth factor-β in tumor microenvironment indicated their role in polarization of TAM towards proinflammatory state. Bone marrow cell of Listeria-administered tumor-bearing mice showed augmented survival, declined expression of p53 upregulated modulator of apoptosis with an upregulated differentiation into activation responsive bone marrow-derived macrophages along with altered expression of macrophage-colony stimulating factor, macrophage-colony stimulating factor receptor, and granulocyte macrophage-colony stimulating factor receptor. These findings indicate that Listeria infection is associated with an augmented differentiation of macrophages accompanied by tumoricidal activation of TAM.

  6. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  7. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction

    Science.gov (United States)

    Chartoff, Elena H.; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we

  8. Sex differences in kappa opioid receptor function and their potential impact on addiction

    Directory of Open Access Journals (Sweden)

    Elena eChartoff

    2015-12-01

    Full Text Available Behavioral, biological and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN, an endogenous ligand at kappa opioid receptors (KORs, is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain,mood and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN gene, genetic linkage with the melanocortin-1 receptor (MC1R, heterodimerization of KORs and mu opioid receptors (MORs, and gonadal hormones

  9. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction.

    Science.gov (United States)

    Chartoff, Elena H; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we

  10. Negative modulation of NMDA receptor channel function by DREAM/calsenilin/KChIP3 provides neuroprotection?

    Science.gov (United States)

    Wang, KeWei; Wang, Yun

    2012-01-01

    N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels highly permeable to calcium and essential to excitatory neurotransmission. The NMDARs have attracted much attention because of their role in synaptic plasticity and excitotoxicity. Evidence has recently accumulated that NMDARs are negatively regulated by intracellular calcium binding proteins. The calcium-dependent suppression of NMDAR function serves as a feedback mechanism capable of regulating subsequent Ca2+ entry into the postsynaptic cell, and may offer an alternative approach to treating NMDAR-mediated excitotoxic injury. This short review summarizes the recent progress made in understanding the negative modulation of NMDAR function by DREAM/calsenilin/KChIP3, a neuronal calcium sensor (NCS) protein. PMID:22518099

  11. Syndecans as cell surface receptors: Unique structure equates with functional diversity

    DEFF Research Database (Denmark)

    Choi, Youngsil; Chung, Heesung; Jung, Heyjung

    2011-01-01

    An increasing number of functions for syndecan cell surface heparan sulfate proteoglycans have been proposed over the last decade. Moreover, aberrant syndecan regulation has been found to play a critical role in multiple pathologies, including cancers, as well as wound healing and inflammation....... As receptors, they have much in common with other molecules on the cell surface. Syndecans are type I transmembrane molecules with cytoplasmic domains that link to the actin cytoskeleton and can interact with a number of regulators. However, they are also highly complex by virtue of their external...... glycosaminoglycan chains, especially heparan sulfate. This heterodisperse polysaccharide has the potential to interact with many ligands from diverse protein families. Here, we relate the structural features of syndecans to some of their known functions....

  12. Investigation of aflatoxin M1 degradation in milk

    Directory of Open Access Journals (Sweden)

    Smajlović Ahmed

    2012-01-01

    Full Text Available Aflatoxin M1 is a highly toxic 4-hydroxylated metabolite of aflatoxins B1 and B2. It is one of the most potent hepatocarcinogens, mutagens, teratogens and immunosuppressors. Feed is often contaminated with aflatoxigenic moulds and aflatoxins with a high possibility of contaminating milk and dairy products with aflatoxin M1. Samples of artificially contaminated milk were exposed to the effects of physical conditions (temperature of -18oC and for microwaves in a microwave oven, time (during the period from 1 to 12 months and a combination of the above mentioned conditions. Following this, levels of aflatoxin M1 degradation were established by using the ELISA method. An insignificant decrease in concentration of toxin was observed which indicates that a temperature of -18°C does not significantly influence the concentration of aflatoxin M1 in the artificially contaminated milk. At the same time, treatment of milk with microwaves in a microwave oven showed an insignificant influence on the percentage of aflatoxin M1 absorbance.

  13. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst(2) and sst(5) receptors) in characteristic proportions. In other words, levels of individual......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  14. Tachykinin NK2 receptor and functional mechanisms in human colon: changes with indomethacin and in diverticular disease and ulcerative colitis.

    Science.gov (United States)

    Burcher, Elizabeth; Shang, Fei; Warner, Fiona J; Du, Qin; Lubowski, David Z; King, Denis W; Liu, Lu

    2008-01-01

    Neurokinin A (NKA) is an important spasmogen in human colon. We examined inflammatory disease-related changes in the tachykinin NK(2) receptor system in human sigmoid colon circular muscle, using functional, radioligand binding, and quantitative reverse transcription-polymerase chain reaction methods. In circular muscle strips, indomethacin enhanced contractile responses to NKA (p diverticular disease (DD) specimens, indicating NK(2) receptor-mediated release of relaxant prostanoids. Contractile responses to both tachykinins were reduced in strips from DD (p disease patients, demonstrating that the change in responsiveness to tachykinins in disease is specifically mediated by the NK(2) receptor. In membranes from UC specimens, receptor affinity for (125)I-NKA (median K(D) 0.91 nM, n = 16) was lower (p disease-related changes in receptor number (B(max)) were found (mean, 2.0-2.5 fmol/mg of wet weight tissue), suggesting that the reduced contractile responses in disease are not due to a loss of receptor number. Different mechanisms may account for the reduced contractility in DD compared with UC. A gender-related difference in receptor density was seen in controls, with B(max) lower in females (1.77 fmol/mg, n = 15) than in males (2.60 fmol/mg, n = 25, p = 0.01). In contrast, no gender-related differences were seen in NK(2) receptor mRNA in control colonic muscle, indicating that the gender difference is a post-translational event.

  15. Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou; Kobe, Bostjan

    2017-04-06

    Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

  16. UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor

    DEFF Research Database (Denmark)

    Cohen, A; Shainberg, Asher; Hochhauser, E

    2011-01-01

    Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5......'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups...... used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group...

  17. Functional μ-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area.

    Science.gov (United States)

    Moreno, Estefanía; Quiroz, César; Rea, William; Cai, Ning-Sheng; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; Ferré, Sergi

    2017-02-01

    The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of μ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin-opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR-Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identified in situ in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, in vivo microdialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders. The μ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallel in vitro experiments in mammalian transfected cells and in situ and in vivo experiments in rat VTA, we demonstrate that a significant population of these MORs form

  18. The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

    Science.gov (United States)

    Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

    2007-09-01

    Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

  19. M1 transitions between superdeformed states in 195Tl

    International Nuclear Information System (INIS)

    Zheng Xing; Xingqu Chen; Xiaochun Wang

    1996-01-01

    Using a triaxial-particle-rotor model, the quadrupole and dipole transition energies, kinematic and dynamic moments of inertia, electromagnetic transition probabilities and the relative intensity of the E2 γ-transitions are calculated for superdeformed bands in 195 Tl. A strong perturbation effect of rotation on transition energies and M1 and E2 transitions of superdeformed states is investigated. The total M1 transitions, enhanced by internal conversion, are expected to compete strongly with the E2 γ-ray at low spins in the superdeformed 195 Tl nucleus. (author)

  20. M1-transitions in the MIT bag model

    International Nuclear Information System (INIS)

    Hackman, R.H.; Deshpande, N.G.; Dicus, D.A.; Teplitz, V.L.

    1977-03-01

    In the MIT bag model, the M1-transitions of low lying hadrons are investigated. The following calculations are performed: 32 hadron masses are recomputed with a choice of bag parameters designed to give the correct values for the proton magnetic moment, μ/sub p/, and several masses, M/sub rho/ M/sub ω/ M/sub Δ/ M/sub Ω/, and M/sub D/; (2) eta, eta', eta/sub c/ mixing is computed in an untrustworthy approximation; and the widths for 38 M1-transitions are computed

  1. Genetic analysis of rice semidwarf mutant Tad-M-1

    International Nuclear Information System (INIS)

    Wang Naiyuan; Yang Rencui

    1995-01-01

    This paper dealed with the inheritance of the rice semidwarf of Tad-M-,a mutant line bred from traditional indica rice Variety Tadukan by radiation. The results indicated that semidwarf of Tad-M-1 was controlled by one pair of recessive gene, which was nonallelic to sd-1 gene of variety Aijiaonante and sd-g gene of variety Xinguiai and allelic to the semidwarf gene of Yunnan japonica variety Xueheaizao and Sichuan indica variety Yizila.The possible uses of Tad-M-1 in rice breeding was also discussed

  2. Methylphenidate and Atomoxetine Enhance Prefrontal Function through alpha[subscript 2]-Adrenergic and Dopamine D[subscript 1] Receptors

    Science.gov (United States)

    Gamo, Nao J.; Wang, Min; Arnsten, Amy F. T.

    2010-01-01

    Objective: This study examined the effects of the attention-deficit/hyperactivity disorder treatments, methylphenidate (MPH) and atomoxetine (ATM), on prefrontal cortex (PFC) function in monkeys and explored the receptor mechanisms underlying enhancement of PFC function at the behavioral and cellular levels. Method: Monkeys performed a working…

  3. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

    Science.gov (United States)

    Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

    1996-08-16

    Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

  4. Multigenerational effects of adolescent morphine exposure on dopamine D2 receptor function.

    Science.gov (United States)

    Byrnes, John J; Johnson, Nicole L; Carini, Lindsay M; Byrnes, Elizabeth M

    2013-05-01

    The use and misuse of prescription opiates in adolescent populations, and in particular, adolescent female populations, has increased dramatically in the past two decades. Given the significant role that opioids play in neuroendocrine function, exposure to opiates during this critical developmental period could have significant consequences for the female, as well as her offspring. In the current set of studies, we utilized the female rat to model the transgenerational impact of adolescent opiate exposure. We examined locomotor sensitization in response to the dopamine D2/D3 receptor agonist quinpirole in the adult male progeny (F1 and F2 generations) of females exposed to morphine during adolescence. All females were drug-free for at least 3 weeks prior to conception, eliminating the possibility of direct fetal exposure to morphine. Both F1 and F2 progeny of morphine-exposed females demonstrated attenuated locomotor sensitization following repeated quinpirole administration. These behavioral effects were coupled with increased quinpirole-induced corticosterone secretion and upregulated kappa opioid receptor and dopamine D2 receptor (D2R) gene expression within the nucleus accumbens. These results suggest significant modifications in response to repeated D2R activation in the progeny of females exposed to opiates during adolescence. Given the significant role that the D2R plays in psychopathology, adolescent opiate exposure could shift the vulnerability of future offspring to psychological disorders, including addiction. Moreover, that effects are also observed in the F2 generation suggests that adolescent opiate exposure can trigger transgenerational epigenetic modifications impacting systems critical for motivated behavior.

  5. Anatomy and behavioral function of serotonin receptors in Drosophila melanogaster larvae.

    Directory of Open Access Journals (Sweden)

    Annina Huser

    Full Text Available The biogenic amine serotonin (5-HT is an important neuroactive molecule in the central nervous system of the majority of animal phyla. 5-HT binds to specific G protein-coupled and ligand-gated ion receptors to regulate particular aspects of animal behavior. In Drosophila, as in many other insects this includes the regulation of locomotion and feeding. Due to its genetic amenability and neuronal simplicity the Drosophila larva has turned into a useful model for studying the anatomical and molecular basis of chemosensory behaviors. This is particularly true for the olfactory system, which is mostly described down to the synaptic level over the first three orders of neuronal information processing. Here we focus on the 5-HT receptor system of the Drosophila larva. In a bipartite approach consisting of anatomical and behavioral experiments we describe the distribution and the implications of individual 5-HT receptors on naïve and acquired chemosensory behaviors. Our data suggest that 5-HT1A, 5-HT1B, and 5-HT7 are dispensable for larval naïve olfactory and gustatory choice behaviors as well as for appetitive and aversive associative olfactory learning and memory. In contrast, we show that 5-HT/5-HT2A signaling throughout development, but not as an acute neuronal function, affects associative olfactory learning and memory using high salt concentration as a negative unconditioned stimulus. These findings describe for the first time an involvement of 5-HT signaling in learning and memory in Drosophila larvae. In the longer run these results may uncover developmental, 5-HT dependent principles related to reinforcement processing possibly shared with adult Drosophila and other insects.

  6. Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors.

    Science.gov (United States)

    Bryant, A H; Menzies, G E; Scott, L M; Spencer-Harty, S; Davies, L B; Smith, R A; Jones, R H; Thornton, C A

    2017-07-01

    The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways, that yield a cytokine output in response to pathogenic stimuli, have been postulated to link infection at the maternal-fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll-like receptors (TLR-3, -7, -8 and -9), and retinoic acid-inducible gene 1 (RIG-I)-like receptors [RIG-I, melanoma differentiation-associated protein 5 (MDA5) and Laboratory of Genetics and Physiology 2(LGP2)] in human term gestation-associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)-6 and/or IL-8 production in response to specific agonists for TLR-3 (Poly(I:C); low and high molecular weight), TLR-7 (imiquimod), TLR-8 (ssRNA40) and RIG-I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR-9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL-8 response, while the choriodecidua and amnion generate a more similar IL-6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR-3, TLR-7, TLR-8 and RIG-1/MDA5 is a broad feature of human term gestation-associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes. © 2017 British Society for Immunology.

  7. Functionalized Ergot-alkaloids as potential dopamine D3 receptor agonists for treatment of schizophrenia

    Science.gov (United States)

    Ivanova, Bojidarka; Spiteller, Michael

    2012-12-01

    The relationship between the molecular structure and physical properties of functionalized naturally occurred Ergot-alkaloids as potential dopamine D3 receptor agonists is presented. The molecular modeling of the ergoline-skeleton is based on the comprehensive theoretical study of the binding affinity of the isolated chemicals towards the active sites of the D3 sub-type receptor (D3R) loops. The studied proton accepting ability under physiological conditions allows classifying four types of monocationics, characterizing with the different binding modes to D3R involving selected amino acid residues to the active sites. These results marked the pharmaceutical potential and clinical usage of the reported compounds as antipsychotic drugs for Schizophrenia treatment, since they allowed evaluating the highlights of the different hypothesizes of the biochemical causes the illness. The applied complex approach for theoretical and experimental elucidation, including quantum chemistry method, electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometric (MS) methods, nuclear magnetic resonance and vibrational IR and Raman spectroscopy on the isolated fifteen novel derivatives (1)-(15) and their different protonated forms (1a)-(15a) evidenced a strong dependence of molecular conformation, physical properties and binding affinity. Thus, the semi-synthetic functionalization of the naturally occurred products (NPs), provided significant possibilities to further molecular drugs-design and development of novel derivatives with wanted biological function, using the established profile of selected classes/families of NPs. The work described chiefly the non-linear (NL) approach for the interpretation of the mass chromatograms on the performed hybrid high performance liquid chromatography (HPLC) tandem MS/MS and MS/MS/MS experiments, discussing the merits and great diversity of instrumentation flexibility, thus achieving fundamental

  8. Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain.

    Science.gov (United States)

    Nation, Kelsey M; De Felice, Milena; Hernandez, Pablo I; Dodick, David W; Neugebauer, Volker; Navratilova, Edita; Porreca, Frank

    2018-05-01

    The response of diffuse noxious inhibitory controls (DNIC) is often decreased, or lost, in stress-related functional pain syndromes. Because the dynorphin/kappa opioid receptor (KOR) pathway is activated by stress, we determined its role in DNIC using a model of stress-induced functional pain. Male, Sprague-Dawley rats were primed for 7 days with systemic morphine resulting in opioid-induced hyperalgesia. Fourteen days after priming, when hyperalgesia was resolved, rats were exposed to environmental stress and DNIC was evaluated by measuring hind paw response threshold to noxious pressure (test stimulus) after capsaicin injection in the forepaw (conditioning stimulus). Morphine priming without stress did not alter DNIC. However, stress produced a loss of DNIC in morphine-primed rats in both hind paws that was abolished by systemic administration of the KOR antagonist, nor-binaltorphimine (nor-BNI). Microinjection of nor-BNI into the right, but not left, central nucleus of the amygdala (CeA) prevented the loss of DNIC in morphine-primed rats. Diffuse noxious inhibitory controls were not modulated by bilateral nor-BNI in the rostral ventromedial medulla. Stress increased dynorphin content in both the left and right CeA of primed rats, reaching significance only in the right CeA; no change was observed in the rostral ventromedial medulla or hypothalamus. Although morphine priming alone is not sufficient to influence DNIC, it establishes a state of latent sensitization that amplifies the consequences of stress. After priming, stress-induced dynorphin/KOR signaling from the right CeA inhibits DNIC in both hind paws, likely reflecting enhanced descending facilitation that masks descending inhibition. Kappa opioid receptor antagonists may provide a new therapeutic strategy for stress-related functional pain disorders.

  9. Dysregulation of chemokine receptor expression and function in leukocytes from ALS patients.

    Science.gov (United States)

    Perner, Caroline; Perner, Florian; Stubendorff, Beatrice; Förster, Martin; Witte, Otto W; Heidel, Florian H; Prell, Tino; Grosskreutz, Julian

    2018-03-28

    Amyotrophic lateral sclerosis (ALS) is rapidly progressive adult-onset motor neuron disease characterized by the neurodegeneration of both upper and lower motor neurons in the cortex and the spinal cord; the majority of patients succumb to respiratory failure. Although the etiology is not yet fully understood, there is compelling evidence that ALS is a multi-systemic disorder, with peripheral inflammation critically contributing to the disease process. However, the full extent and nature of this immunological dysregulation remains to be established, particularly within circulating blood cells. Therefore, the aim of the present study was to identify dysregulated inflammatory molecules in peripheral blood cells of ALS patients and analyze for functional consequences of the observed findings. To this end, we employed flow cytometry-based screening to quantify the surface expression of major chemokine receptors and integrins. A significantly increased expression of CXCR3, CXCR4, CCL2, and CCL5 was observed on T cells in ALS patients compared to healthy controls. Intriguingly, the expression was even more pronounced in patients with a slow progressive phenotype. To further investigate the functional consequences of this altered surface expression, we used a modified Boyden chamber assay to measure chemotaxis in ALS patient-derived lymphocytes. Interestingly, chemoattraction with the CXCR3-Ligand IP10 led to upregulated migratory behavior of ALS lymphocytes compared to healthy controls. Taken together, our data provides evidence for a functional dysregulation of IP10-directed chemotaxis in peripheral blood cells in ALS patients. However, whether the chemokine itself or its receptor CXCR3, or both, could serve as potential therapeutic targets in ALS requires further investigations.

  10. CD64: An Attractive Immunotherapeutic Target for M1-type Macrophage Mediated Chronic Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Olusiji A. Akinrinmade

    2017-09-01

    Full Text Available To date, no curative therapy is available for the treatment of most chronic inflammatory diseases such as atopic dermatitis, rheumatoid arthritis, or autoimmune disorders. Current treatments require a lifetime supply for patients to alleviate clinical symptoms and are unable to stop the course of disease. In contrast, a new series of immunotherapeutic agents targeting the Fc γ receptor I (CD64 have emerged and demonstrated significant clinical potential to actually resolving chronic inflammation driven by M1-type dysregulated macrophages. This subpopulation plays a key role in the initiation and maintenance of a series of chronic diseases. The novel recombinant M1-specific immunotherapeutics offer the prospect of highly effective treatment strategies as they have been shown to selectively eliminate the disease-causing macrophage subpopulations. In this review, we provide a detailed summary of the data generated, together with the advantages and the clinical potential of CD64-based targeted therapies for the treatment of chronic inflammatory diseases.

  11. Design and optimization of selective azaindole amide M1 positive allosteric modulators.

    Science.gov (United States)

    Davoren, Jennifer E; O'Neil, Steven V; Anderson, Dennis P; Brodney, Michael A; Chenard, Lois; Dlugolenski, Keith; Edgerton, Jeremy R; Green, Michael; Garnsey, Michelle; Grimwood, Sarah; Harris, Anthony R; Kauffman, Gregory W; LaChapelle, Erik; Lazzaro, John T; Lee, Che-Wah; Lotarski, Susan M; Nason, Deane M; Obach, R Scott; Reinhart, Veronica; Salomon-Ferrer, Romelia; Steyn, Stefanus J; Webb, Damien; Yan, Jiangli; Zhang, Lei

    2016-01-15

    Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Placental Chemokine Receptor D6 Is Functionally Impaired in Pre-Eclampsia.

    Directory of Open Access Journals (Sweden)

    Chiara Tersigni

    Full Text Available Pre-eclampsia (PE is a major cause of maternal and perinatal morbidity and mortality worldwide. It is defined by new onset of hypertension and proteinuria after the 20th week of gestation and characterized by systemic exaggerated inflammatory response. D6 is a chemokines scavenger receptor that binds with high affinity CC chemokines, internalizes and targets the ligands for degradation. It is expressed in trophoblast-derived tissues and prevents excessive placenta leukocyte infiltration.The aim of this study was to investigate the expression and function of D6 in human placentae from pre-eclamptic and healthy pregnant women.Plasma levels of D6-binding CC chemokines (CCL-2, CCL-3, CCL-4, CCL-7, CCL-11 and pro-inflammatory cytokines (IL-6, TNF-α, CRP were analyzed in 37 healthy pregnant women and 38 patients with PE by multiplex bead assay. Higher circulating levels of CCL7, CCL11, IL-6, (p<0.0001 and CRP (p<0.05 were observed in PE women compared to controls. Levels of circulating CCL4 were decreased in PE (p<0.001, while no significant differences of CCL2, CCL3 or TNF-α levels were detected. Immunofluorescent staining of placental sections showed higher expression of D6 receptor in the PE syncytiotrophoblast. Confocal and Western blot (WB analyses revealed a prevalent distribution of D6 in trophoblast cells membranes in PE. Increased activation of D6 intracellular pathway was observed by Western blot analyses of p-LIMK and p-cofilin in trophoblast cell lysates. D6 functional assays showed reduced scavenging of CCL2 in PE cells compared to controls. Since actin filaments spatial assembling is essential for D6 intracellular trafficking and scavenging activity, we investigated by confocal microscopy trophoblast cytoskeleton organization and we observed a dramatic disarrangement in PE compared to controls.our results suggest membrane distribution of D6 receptor on trophoblast cell membranes in PE, together with reduced functionality, probably due

  13. assessing aflatoxin m1 levels among lactating mothers' in damaturu ...

    African Journals Online (AJOL)

    userpc

    1Department of Microbiology Ahmadu Bello University Zaria, Kaduna State. ... Aflatoxin M1 (AFM1) is a biomarker of aflatoxin B1 exposure in breast milk, a possible risk factor for infant early ..... Mycotoxins in food and feed: present status and ...

  14. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  15. The melanocortin receptor agonist NDP-MSH impairs the allostimulatory function of dendritic cells.

    Science.gov (United States)

    Rennalls, La'Verne P; Seidl, Thomas; Larkin, James M G; Wellbrock, Claudia; Gore, Martin E; Eisen, Tim; Bruno, Ludovica

    2010-04-01

    As alpha-melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells and has potent immunosuppressive properties, it was determined whether human dendritic cells (DCs) express the receptor for this hormone. Reverse transcription-polymerase chain reaction detected messenger RNA specific for all of the known melanocortin receptors in DCs. Mixed lymphocyte reactions also revealed that treatment with [Nle(4), DPhe(7)]-alpha-MSH (NDP-MSH), a potent alpha-MSH analogue, significantly reduced the ability of DCs to stimulate allogeneic T cells. The expression of various cell surface adhesion, maturation and costimulatory molecules on DCs was also investigated. Although treatment with NDP-MSH did not alter the expression of CD83 and major histocompatibility complex class I and II, the surface expression of CD86 (B7.2), intercellular adhesion molecule (ICAM-1/CD54) and CD1a was reduced. In summary, our data indicate that NDP-MSH inhibits the functional activity of DCs, possibly by down-regulating antigen-presenting and adhesion molecules and that these events may be mediated via the extracellular signal-regulated kinase 1 and 2 pathway.

  16. Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices

    Science.gov (United States)

    Singha, Santiswarup; Shao, Kun; Yang, Yang; Clemente-Casares, Xavier; Solé, Patricia; Clemente, Antonio; Blanco, Jesús; Dai, Qin; Song, Fayi; Liu, Shang Wan; Yamanouchi, Jun; Umeshappa, Channakeshava Sokke; Nanjundappa, Roopa Hebbandi; Detampel, Pascal; Amrein, Matthias; Fandos, César; Tanguay, Robert; Newbigging, Susan; Serra, Pau; Khadra, Anmar; Chan, Warren C. W.; Santamaria, Pere

    2017-07-01

    We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

  17. Expression and function of NOD-like receptors by human term gestation-associated tissues.

    Science.gov (United States)

    Bryant, Aled H; Bevan, Ryan J; Spencer-Harty, Samantha; Scott, Louis M; Jones, Ruth H; Thornton, Catherine A

    2017-10-01

    Nucleotide-binding oligomerization domain (NOD)-like receptors or NOD-like receptors (NLRs) have been implicated in several disease pathologies associated with inflammation. Since local and systemic inflammation is a hallmark of both term and preterm labour, a role for NLRs at the materno-fetal interface has been postulated. Gene expression and immunolocalisation of NLR family members in human placenta, choriodecidua, and amnion were examined. Tissue explants were used to examine the response to activators of NOD1 (Tri-DAP), NOD2 (MDP) and NLRP3 (nigericin). Cell/tissue-free supernatants were examined for the production of interleukin (IL)-1β, IL-6, IL-8 and IL-10 using specific ELISAs. Expression of transcripts for NOD1, NOD2, NLRP3, NLRC4, NLRX1, NLRP1 and NAIP and protein expression of NOD1, NOD2 and NLRP3 were a broad feature of all term gestation-associated tissues. Production of cytokines was increased significantly in response to all ligands in placenta and choriodecidua, except for MDP-induced IL-10. Similarly, there was a significant in the amnion except for MDP induced IL-1β and IL-10 response to either agonist. IL-1β production was dependent on caspase-1 regardless of agonist used or tissue examined. Term human gestation-associated tissues express functional NLRs which likely play a role in both sterile and pathogen-driven inflammatory responses at the materno-fetal interface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Cerebellar nicotinic cholinergic receptors are intrinsic to the cerebellum: implications for diverse functional roles.

    Science.gov (United States)

    Turner, Jill R; Ortinski, Pavel I; Sherrard, Rachel M; Kellar, Kenneth J

    2011-12-01

    Although recent studies have delineated the specific nicotinic subtypes present in the mammalian cerebellum, very little is known about their location or function within the cerebellum. This is of increased interest since nicotinic receptors (nAChRs) in the cerebellum have recently been implicated in the pathology of autism spectrum disorders. To begin to better understand the roles of these heteromeric nAChRs in the cerebellar circuitry and their therapeutic potential as targets for drug development, we used various chemical and stereotaxic lesion models in conjunction with slice electrophysiology to examine how specific heteromeric nAChR subtypes may influence the surrounding cerebellar circuitry. Using subunit-specific immunoprecipitation of radiolabeled nAChRs in the cerebella following N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, p-chloroamphetamine, and pendunculotomy lesions, we show that most, if not all, cerebellar nicotinic receptors are present in cells within the cerebellum itself and not in extracerebellar afferents. Furthermore, we demonstrate that the β4-containing, but not the β2-containing, nAChRs intrinsic to the cerebellum can regulate inhibitory synaptic efficacy at two major classes of cerebellar neurons. These tandem findings suggest that nAChRs may present a potential drug target for disorders involving the cerebellum.

  19. Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors.

    Science.gov (United States)

    Bradshaw, M S; Tsai, S Y; Leng, X H; Dobson, A D; Conneely, O M; O'Malley, B W; Tsai, M J

    1991-09-05

    Steroid response elements (SREs) cooperate with many different cis-acting elements including NF-1 sites, CACCC boxes, and other SREs to induce target gene expression (Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90; Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395). Induction of gene expression can be additive or synergistic with respect to the level of activation by either transactivators. Two mechanisms have been proposed for how synergism occurs: 1) cooperative binding of transcriptional activators to DNA or 2) simultaneous interaction of individually bound activators with a common target protein. We have shown previously that cooperative binding of receptors is important for synergism between two progesterone response elements (PREs). Here we showed that an estrogen response element (ERE) and a PRE can also functionally cooperate and this synergism between an ERE and a PRE is not contributed by cooperative DNA binding. Furthermore, we have demonstrated that the activation domains of the progesterone receptor (PR) (C1Act) are required for synergism between two PREs and sufficient for confirming cooperative binding. However these two activation domains of PR are not sufficient for synergism between an ERE and a PRE. Additional regions within the NH2-terminal and COOH-terminal domains are also required for synergistic interaction between two heterologous SREs.

  20. Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

    Directory of Open Access Journals (Sweden)

    Shweta Joshi

    Full Text Available Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

  1. Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

    Directory of Open Access Journals (Sweden)

    Sierralta Walter

    2009-01-01

    Full Text Available Abstract Background Mating changes the mode of action of 17beta-estradiol (E2 to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER are required. This change was designated as intracellular path shifting (IPS. Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta in oviductal epithelial cells of rats on day 1 of cycle (C1 or pregnancy (P1 using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb and calbindin 9 kDa (s100g in rats on C1 or P1 treated with selective agonists for ESR1 (PPT or ESR2 (DPN. The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

  2. Adenosine A2A receptors and A2A receptor heteromers as key players in striatal function

    Directory of Open Access Journals (Sweden)

    Sergi eFerre

    2011-06-01

    Full Text Available A very significant density of adenosine adenosine A2A receptors (A2ARs is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs. In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs. In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striato-pallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl-cyclase (AC. Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striato-pallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders.

  3. Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum*

    Science.gov (United States)

    Contreras, Estefanía; Schoppmeier, Michael; Real, M. Dolores; Rausell, Carolina

    2013-01-01

    Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders. PMID:23645668

  4. Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

    Science.gov (United States)

    Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

    2008-12-01

    Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

  5. Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

    International Nuclear Information System (INIS)

    Mak, Paul; Jaggi, Meena; Syed, Viqar; Chauhan, Subhash C.; Hassan, Sazzad; Biswas, Helal; Balaji, K.C.

    2008-01-01

    Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells

  6. Functional Characterization of CCHamide and Muscarinic Acetylcholine Receptor Signalling in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Ren, Guilin Robin

    G-protein coupled receptors (GPCRs) constitute a large and ancient superfamily of membraneproteins responsible for the transduction of extracellular signals to the inside of the cells. In thisPh.D. thesis, Drosophila melanogaster (Dm) was used as a model organism to investigate a numberof topics...... is a newly discovered insect peptide hormone. The function of this novel peptide hasnot been well characterised. In this Ph.D. thesis, I identified CCHamide-2 peptides in endocrinecells of the gut and neurones of the brain of larvae and endocrine cells of the gut of adultDrosophila. Behavioural assays...... little is known about muscarinic acetylcholine receptorsignalling in insects. In this study, I found that two types of mAChRs occur in D. melanogaster, onecoupling to Gq (A-type) and the other to Gi (B-type). Both A- and B-type Dm-mAChRs can beactivated by acetylcholine (ACh), but the classical...

  7. Transient receptor potential channel superfamily: Role in lower urinary tract function.

    Science.gov (United States)

    Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

    2015-11-01

    Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

  8. Striatal Dopamine D2/D3 Receptor Availability Is Associated with Executive Function in Healthy Controls but Not Methamphetamine Users.

    Directory of Open Access Journals (Sweden)

    Michael E Ballard

    Full Text Available Dopamine D2/D3 receptor availability in the striatum has been linked with executive function in healthy individuals, and is below control levels among drug addicts, possibly contributing to diminished executive function in the latter group. This study tested for an association of striatal D2/D3 receptor availability with a measure of executive function among research participants who met DSM-IV criteria for methamphetamine dependence.Methamphetamine users and non-user controls (n = 18 per group completed the Wisconsin Card Sorting Test and positron emission tomography with [18F]fallypride.The methamphetamine users displayed significantly lower striatal D2/D3 receptor availability on average than controls after controlling for age and education (p = 0.008, but they did not register greater proportions of either perseverative or non-perseverative errors when controlling for education (both ps ≥ 0.622. The proportion of non-perseverative, but not perseverative, errors was negatively correlated with striatal D2/D3 receptor availability among controls (r = -0.588, p = 0.010, but not methamphetamine users (r = 0.281, p = 0.258, and the group-wise interaction was significant (p = 0.030.These results suggest that cognitive flexibility, as measured by perseverative errors on the Wisconsin Card Sorting Test, is not determined by signaling through striatal D2/D3 receptors in healthy controls, and that in stimulant abusers, who have lower D2/D3 receptor availability, compensation can effectively maintain other executive functions, which are associated with D2/D3 receptor signaling in controls.

  9. Complement Receptor 3 Has Negative Impact on Tumor Surveillance through Suppression of Natural Killer Cell Function

    Directory of Open Access Journals (Sweden)

    Cheng-Fei Liu

    2017-11-01

    Full Text Available Complement receptor 3 (CR3 is expressed abundantly on natural killer (NK cells; however, whether it plays roles in NK cell-dependent tumor surveillance is largely unknown. Here, we show that CR3 is an important negative regulator of NK cell function, which has negative impact on tumor surveillance. Mice deficient in CR3 (CD11b−/− mice exhibited a more activated NK phenotype and had enhanced NK-dependent tumor killing. In a B16-luc melanoma-induced lung tumor growth and metastasis model, mice deficient in CR3 had reduced tumor growth and metastases, compared with WT mice. In addition, adaptive transfer of NK cells lacking CR3 (into NK-deficient mice mediated more efficient suppression of tumor growth and metastases, compared with the transfer of CR3 sufficient NK cells, suggesting that CR3 can impair tumor surveillance through suppression of NK cell function. In vitro analyses showed that engagement of CR3 with iC3b (classical CR3 ligand on NK cells negatively regulated NK cell activity and effector functions (i.e. direct tumor cell killing, antibody-dependent NK-mediated tumor killing. Cell signaling analyses showed that iC3b stimulation caused activation of Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1 and JNK, and suppression of ERK in NK cells, supporting that iC3b mediates negative regulation of NK cell function through its effects on SHIP-1, JNK, and ERK signal transduction pathways. Thus, our findings demonstrate a previously unknown role for CR3 in dysregulation of NK-dependent tumor surveillance and suggest that the iC3b/CR3 signaling is a critical negative regulator of NK cell function and may represent a new target for preserving NK cell function in cancer patients and improving NK cell-based therapy.

  10. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

    Science.gov (United States)

    Walsh, Matthew C; Lee, JangEun; Choi, Yongwon

    2015-07-01

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factor-β receptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Tumor necrosis factor receptor associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system

    Science.gov (United States)

    Walsh, Matthew C.; Lee, JangEun; Choi, Yongwon

    2016-01-01

    Summary Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of IL-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the toll-like receptor (TLR) family, tumor growth factor-β receptors (TGFβR), and T cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor (IRF) pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system, but also for maintaining immune tolerance, and more recent works have begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. PMID:26085208

  12. Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs

    Directory of Open Access Journals (Sweden)

    Westmoreland Susan V

    2010-02-01

    Full Text Available Abstract Background The trace amine associated receptor family is a diverse array of GPCRs that arose before the first vertebrates walked on land. Trace amine associated receptor 1 (TAAR1 is a wide spectrum aminergic receptor that acts as a modulator in brain monoaminergic systems. Other trace amine associated receptors appear to relate to environmental perception and show a birth-and-death pattern in mammals similar to olfactory receptors. Results Across mammals, avians, and amphibians, the TAAR1 gene is intact and appears to be under strong purifying selection based on rates of amino acid fixation compared to neutral mutations. We have found that in dogs it has become a pseudogene. Our analyses using a comparative genetics approach revealed that the pseudogenization event predated the emergence of the Canini tribe rather than being coincident with canine domestication. By assessing the effects of the TAAR1 agonist β-phenylethylamine on [3H]dopamine uptake in canine striatal synaptosomes and comparing the degree and pattern of uptake inhibition to that seen in other mammals, including TAAR1 knockout mice, wild type mice and rhesus monkey, we found that the TAAR1 pseudogenization event resulted in an uncompensated loss of function. Conclusion The gene family has seen expansions among certain mammals, notably rodents, and reductions in others, including primates. By placing the trace amine associated receptors in an evolutionary context we can better understand their function and their potential associations with behavior and neurological disease.

  13. First cytoplasmic loop of glucagon-like peptide-1 receptor can function at the third cytoplasmic loop position of rhodopsin.

    Science.gov (United States)

    Yamashita, Takahiro; Tose, Koji; Shichida, Yoshinori

    2008-01-01

    G protein-coupled receptors (GPCRs) are classified into several families based on their amino acid sequences. In family 1, GPCRs such as rhodopsin and adrenergic receptor, the structure-function relationship has been extensively investigated to demonstrate that exposure of the third cytoplasmic loop is essential for selective G protein activation. In contrast, much less is known about other families. Here we prepared chimeric mutants between Gt-coupled rhodopsin and Gi/Go- and Gs-coupled glucagon-like peptide-1 (GLP-1) receptor of family 2 and tried to identify the loop region that functions at the third cytoplasmic loop position of rhodopsin. We succeeded in expressing a mutant having the first cytoplasmic loop of GLP-1 receptor and found that this mutant activated Gi and Go efficiently but did not activate Gt. Moreover, the rhodopsin mutant having the first loop of Gs-coupled secretin receptor of family 2 decreased the Gi and Go activation efficiencies. Therefore, the first loop of GLP-1 receptor would share a similar role to the third loop of rhodopsin in G protein activation. This result strongly suggested that different families of GPCRs have maintained molecular architectures of their ancestral types to generate a common mechanism, namely exposure of the cytoplasmic loop, to activate peripheral G protein.

  14. Dopamine D(3) receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: role of hyperthermia.

    Science.gov (United States)

    Baladi, Michelle G; Newman, Amy H; Nielsen, Shannon M; Hanson, Glen R; Fleckenstein, Annette E

    2014-06-05

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Dopamine D3 receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: Role of hyperthermia

    Science.gov (United States)

    Baladi, Michelle G.; Newman, Amy H.; Nielsen, Shannon M.; Hanson, Glen R.; Fleckenstein, Annette E.

    2014-01-01

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. PMID:24685638

  16. Molecular Evolution and Functional Divergence of Trace Amine-Associated Receptors.

    Directory of Open Access Journals (Sweden)

    Seong-Il Eyun

    Full Text Available Trace amine-associated receptors (TAARs are a member of the G-protein-coupled receptor superfamily and are known to be expressed in olfactory sensory neurons. A limited number of molecular evolutionary studies have been done for TAARs so far. To elucidate how lineage-specific evolution contributed to their functional divergence, we examined 30 metazoan genomes. In total, 493 TAAR gene candidates (including 84 pseudogenes were identified from 26 vertebrate genomes. TAARs were not identified from non-vertebrate genomes. An ancestral-type TAAR-like gene appeared to have emerged in lamprey. We found four therian-specific TAAR subfamilies (one eutherian-specific and three metatherian-specific in addition to previously known nine subfamilies. Many species-specific TAAR gene duplications and losses contributed to a large variation of TAAR gene numbers among mammals, ranging from 0 in dolphin to 26 in flying fox. TAARs are classified into two groups based on binding preferences for primary or tertiary amines as well as their sequence similarities. Primary amine-detecting TAARs (TAAR1-4 have emerged earlier, generally have single-copy orthologs (very few duplication or loss, and have evolved under strong functional constraints. In contrast, tertiary amine-detecting TAARs (TAAR5-9 have emerged more recently and the majority of them experienced higher rates of gene duplications. Protein members that belong to the tertiary amine-detecting TAAR group also showed the patterns of positive selection especially in the area surrounding the ligand-binding pocket, which could have affected ligand-binding activities and specificities. Expansions of the tertiary amine-detecting TAAR gene family may have played important roles in terrestrial adaptations of therian mammals. Molecular evolution of the TAAR gene family appears to be governed by a complex, species-specific, interplay between environmental and evolutionary factors.

  17. Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

    Science.gov (United States)

    Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

    2008-01-01

    Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

  18. Molecular cloning, functional characterization, and evolutionary analysis of vitamin D receptors isolated from basal vertebrates.

    Directory of Open Access Journals (Sweden)

    Erin M Kollitz

    Full Text Available The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus, a 1R jawless fish; the little skate (Leucoraja erinacea, a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus, a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may

  19. Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

    Science.gov (United States)

    Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

    2017-09-01

    We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4 + T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4 + T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4 + T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4 + T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4 + T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4 + T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Functional hypothalamic amenorrhea due to increased CRH tone in melanocortin receptor 2-deficient mice.

    Science.gov (United States)

    Matsuwaki, Takashi; Nishihara, Masugi; Sato, Tsuyoshi; Yoda, Tetsuya; Iwakura, Yoichiro; Chida, Dai

    2010-11-01

    Exposure to chronic stressors results in dysregulation of the hypothalamic-pituitary-adrenal axis and a disruption in reproduction. CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis induces the secretion of ACTH from the pituitary, which stimulates adrenal steroidogenesis via the specific cell-surface melanocortin 2 receptor (MC2R). Previously, we demonstrated that MC2R(-/-) mice had undetectable levels of corticosterone despite high ACTH levels. Here, we evaluated the reproductive functions of female MC2R(-/-) mice and analyzed the mechanism of the disrupted cyclicity of these mice. The expression of CRH in the paraventricular nucleus was significantly increased in MC2R(-/-) mice under nonstressed conditions. Although MC2R(-/-) females were fertile, they showed a prolonged estrous cycle. After hormonal stimulation, MC2R(-/-) females produced nearly-normal numbers of eggs, but slightly less than MC2R(+/-) females, and showed near-normal ovarian histology. During diestrus, the number of GnRH-positive cells in the medial preoptic area was significantly reduced in MC2R(-/-) females. CRH type 1 receptor antagonist restored estrous cyclicity in MC2R(-/-) females. Kisspeptin-positive areas in the arcuate nucleus were comparable, whereas kisspeptin-positive areas in the anteroventral periventricular nucleus in MC2R(-/-) females were significantly reduced compared with MC2R(+/-) females, suggesting that arcuate nucleus kisspeptin is not involved, but anteroventral periventricular nucleus kisspeptin may be involved, in the maintenance of estrous cyclicity. Our findings show that high levels of hypothalamic CRH disturb estrous cyclicity in the female animals and that the MC2R(-/-) female is a unique animal model of functional hypothalamic amenorrhea.

  1. Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA.

    Directory of Open Access Journals (Sweden)

    Vijay R Gupta

    2008-05-01

    Full Text Available The vacuolating cytotoxin (VacA of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM, as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.

  2. HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection.

    Science.gov (United States)

    Costafreda, Maria Isabel; Kaplan, Gerardo

    2018-05-01

    The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV. IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the

  3. Learning stage-dependent effect of M1 disruption on value-based motor decisions.

    Science.gov (United States)

    Derosiere, Gerard; Vassiliadis, Pierre; Demaret, Sophie; Zénon, Alexandre; Duque, Julie

    2017-11-15

    The present study aimed at characterizing the impact of M1 disruption on the implementation of implicit value information in motor decisions, at both early stages (during reinforcement learning) and late stages (after consolidation) of action value encoding. Fifty subjects performed, over three consecutive days, a task that required them to select between two finger responses according to the color (instruction) and to the shape (implicit, undisclosed rule) of an imperative signal: considering the implicit rule in addition to the instruction allowed subjects to earn more money. We investigated the functional contribution of M1 to the implementation of the implicit rule in subjects' motor decisions. Continuous theta burst stimulation (cTBS) was applied over M1 either on Day 1 or on Day 3, producing a temporary lesion either during reinforcement learning (cTBS Learning group) or after consolidation of the implicit rule, during decision-making (cTBS Decision group), respectively. Interestingly, disrupting M1 activity on Day 1 improved the reliance on the implicit rule, plausibly because M1 cTBS increased dopamine release in the putamen in an indirect way. This finding corroborates the view that cTBS may affect activity in unstimulated areas, such as the basal ganglia. Notably, this effect was short-lasting; it did not persist overnight, suggesting that the functional integrity of M1 during learning is a prerequisite for the consolidation of implicit value information to occur. Besides, cTBS over M1 did not impact the use of the implicit rule when applied on Day 3, although it did so when applied on Day 2 in a recent study where the reliance on the implicit rule declined following cTBS (Derosiere et al., 2017). Overall, these findings indicate that the human M1 is functionally involved in the consolidation and implementation of implicit value information underlying motor decisions. However, M1 contribution seems to vanish as subjects become more experienced in using

  4. Expression and function of proton-sensing G-protein-coupled receptors in inflammatory pain

    Directory of Open Access Journals (Sweden)

    Lin Chih-Shin

    2009-07-01

    Full Text Available Abstract Background Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs are expressed in pain-relevant loci (dorsal root ganglia, DRG, which suggests their possible involvement in nociception, but their functions in pain remain unclear. Results In this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freund's adjuvant (CFA. In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1 was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function. Conclusion Each subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after

  5. DMPD: CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multipleligand specificities and functions. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available ) (.html) (.csml) Show CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multipleligand specificities and function...d NK cell membrane receptor with multipleligand specificities and functions. Authors Ross GD, Vetvicka V. Pu...igand specificities and functions. Ross GD, Vetvicka V. Clin Exp Immunol. 1993 May;92(2):181-4. (.png) (.svg...8485905 CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiplel

  6. Modulation of NMDA receptor function by ketamine and magnesium: Part I

    NARCIS (Netherlands)

    Liu, H. T.; Hollmann, M. W.; Liu, W. H.; Hoenemann, C. W.; Durieux, M. E.

    2001-01-01

    N-methyl-D-aspartate (NMDA) receptors are important components of pain processing. Ketamine and Mg2+ block NMDA receptors and might therefore be useful analgesics, and combinations of Mg2+ and ketamine provide more effective analgesia. We investigated their interactions at NMDA receptors. Xenopus

  7. The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

    NARCIS (Netherlands)

    Schmitz, Leanne J M; Klaassen, Remco V; Ruiperez-Alonso, Marta; Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J; Mansvelder, Huib D; Smit, August B; Spijker, Sabine; Verhage, Matthijs

    2017-01-01

    Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with

  8. A critical look at the function of the P2Y11 receptor

    DEFF Research Database (Denmark)

    Dreisig, Karin; Kornum, Birgitte Rahbek

    2016-01-01

    The P2Y11 receptor is a member of the purinergic receptor family. It has been overlooked, somewhat due to the lack of a P2ry11 gene orthologue in the murine genome, which prevents the generation of knockout mice, which have been so helpful for defining the roles of other P2Y receptors. Furthermor...

  9. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

    DEFF Research Database (Denmark)

    Voldborg, B R; Damstrup, L; Spang-Thomsen, M

    1997-01-01

    The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intr...... aspects of therapeutic targeting of EGFR....

  10. The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria

    NARCIS (Netherlands)

    Fabriek, Babs O.; van Bruggen, Robin; Deng, Dong Mei; Ligtenberg, Antoon J. M.; Nazmi, Kamran; Schornagel, Karin; Vloet, Rianka P. M.; Dijkstra, Christine D.; van den Berg, Timo K.

    2009-01-01

    The plasma membrane glycoprotein receptor CD163 is a member of the scavenger receptor cystein-rich (SRCR) superfamily class B that is highly expressed on resident tissue macrophages in vivo. Previously, the molecule has been shown to act as a receptor for hemoglobin-haptoglobin complexes and to

  11. M1 and E2 transitions in the ground-state configuration of atomic ...

    Indian Academy of Sciences (India)

    have calculated the forbidden transition (M1 and E2) parameters such as transition energies, log- arithmic weighted ... Keywords. Forbidden transitions; transition energies; logarithmic weighted oscillator strengths; .... optimizing the energy function based on the non-relativistic Hamiltonian of an atom,. HNR = N. ∑ j=1. (12∇ ...

  12. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

    Science.gov (United States)

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-03-06

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase.

  13. The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

    Directory of Open Access Journals (Sweden)

    Sara B Estruch

    Full Text Available Nuclear orphan receptor TLX (NR2E1 functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1, a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9, a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

  14. The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

    Science.gov (United States)

    Estruch, Sara B; Buzón, Víctor; Carbó, Laia R; Schorova, Lenka; Lüders, Jens; Estébanez-Perpiñá, Eva

    2012-01-01

    Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

  15. Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

    Science.gov (United States)

    Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

    2002-09-01

    Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the sa